Dual polarity analysis of nucleic acids

ABSTRACT

This invention provides methods for characterizing the amounts of nucleic acids, including plus/minus determinations, the use of different constructs, the use of a library and a reference library. Expression may also be compared in two or more samples using the methods of this invention. Also provided are heterophasic arrays comprising labeled positive copies of nucleic acids hybridized to the array and labeled negative copies of nucleic acids hybridized to the array, in which the labeled positive copies are separately quantifiable from the labeled negative copies.

CROSS-REFERENCE TO OTHER RELATED APPLICATIONS

This application claims the benefit of U.S. Patent Application20050137388 (published on Jun. 23, 2005), accorded Ser. No. 096076(filed on Mar. 12, 2002), and U.S. Patent Application 20060057583(published on Mar. 16, 2006), accorded Ser. No. 693481 (filed on Oct.24, 2003), the latter application being a continuation-in part of U.S.Patent Application 20040161741 (published on Aug. 19, 2004), Ser. No.896897 (filed on Jun. 30, 2001). The contents of the aforementionedapplications are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

This invention relates to the field of nucleic acid detection, includingthe quantitative determination and characterization of unknown nucleicacids in a sample using an array format.

All patents, patent applications, patent publications, scientificarticles and the like, cited or identified in this application arehereby incorporated by reference in their entirety in order to describemore fully the state of the art to which the present invention pertains.

BACKGROUND OF THE INVENTION

The use of arrays to simultaneously quantify a large number of nucleicacid targets in a single experimental sample is an increasingly popularmethod. There are two areas where this method is most widely used. Firstis the generation of a mRNA profile to examine effects of differentconditions (genetic or environmental) on mRNA expression. Second is thegeneration of a gene dosage profile to examine the presence ofamplifications or deletions of portions of genomic DNA (comparativegenome hybridization, CGH).

In the first area, labeled copies (cDNA) have been made from mRNAtemplates by reverse transcription, or less commonly the mRNA itself hasbeen directly labeled. For examples of the latter, psoralen-biotin(Kumar et al., 2002 Nature Biotechnology 20; 58-63; incorporated byreference herein) and a ligation reaction (Kampa et al., 2002 GenomeResearch 14; 331-342; incorporated by reference herein) have been usedto label purified poly A RNA in studies where there were concerns aboutavoiding potential artifacts caused by copying reactions.

In the second area involving CGH studies, labeled genomic DNA has beenprepared through a nick translation or random primer reaction. Analternative method has been to directly label the genomic DNA itselfwith chemical reagents. Signals from a test sample can be compared to astandard to indicate the presence of increases or decreases in eithergenetic representation (CGH) or expression (RNA profiling) of variousnucleic acid sequences. The standard can either be done simultaneouslyor in parallel with the test sample or the standard can even compriseprior or archived data. In many cases, the standard will be a controlsample: cells growing under “normal” conditions vs some environmentalfactor or it can be a transformed cell versus an untransformed cell. Inother cases, the standard is of an arbitrary nature, such as in thecase, for example, where kidney cell expression is measured and comparedto liver cell expression as a reference standard, thereby identifyinggenes that have differential expression in kidneys versys liver. Inanother example, lung cancer can be compared to normal lung cells andbreast cancer cells and the latter two can serve as reference standards.

In either RNA profiling or CGH array applications, hybridization of thelabeled products then takes place with complementary nucleic acidslocated at various sites on the array followed by quantification of theamount of signal strength at each location. The strands on each site ofthe array can be single strands comprising synthetic oligonucleotides orpolynucleotides that represent a selected portion of the nucleic acidsequence of interest (a monophasic array), or the strands may be derivedfrom denatured double-stranded sources such as bacterial artificialchromosomes (BACs), plasmids or PCR products (biphasic arrays). In thelatter case, when labeled mRNA or cDNA were used as probes for mRNAprofiling, only one strand has usually served as a target even thoughboth strands are present at each site on the biphasic array.

There are numerous situations, however, where the sample size isinsufficient to produce effective amounts of signals on an array and theamount of nucleic acids in the sample needs to be amplified. In thefirst method that was designed for global amplification of mRNA (anddescribed as the Eberwine process by Van Gelder et al. (1990, Proc.Natl. Acad. Sci. USA 87; 1663-1667, incorporated by reference herein)),a primer with a T7 promoter attached to an oligo-T segment was used toprepare cDNA copies by extension from the poly A region of mRNA togenerate a hybrid molecule with the cDNA bound to its complementary mRNAtemplate. In a subsequent step, the method of Gubler and Hoffman (1983Gene 25; 263-269, incorporated by reference herein) was used to allowportions of the original template mRNA to be used as primers, therebytransforming the original first strand cDNA copies into double-strandedDNA constructs. Because the T7 promoter sequence was included in theoriginal oligo-T primer, the second strand synthesis step converts thisprimer segment into a functional double stranded-promoter that can beused in a transcription reaction for synthesis of a large number of RNAcopies from each DNA template. Unlike the original mRNA which had a polyA segment at the 3′ end, the RNA copies made by this amplificationmethod have a poly T sequence at the 5′ end, i.e., the RNA copies arethe opposite strand of the original mRNA and are sometimes referred toas aRNA. As described previously for labeled cDNA, the labeled aRNAcreated from the Eberwine process has been used with arrays that haveeither both strands present (a biphasic array) or have targets with theoriginal mRNA sequences (a monophasic array).

Although this orientation for constructs to make a labeled RNA libraryis the most common, other methods have been described where abacteriophage promoter is incorporated into the other end, i.e., thetranscription takes place in nucleic acid constructs from the end of thenucleic acid constructs that was derived from the original 5′ end ofmRNAs, thereby generating sense RNA that is essentially similar to theoriginal starting mRNA. Instead of using the endogenous mRNA templatesas a source of primers as described by Eberwine et al., this othermethod uses an exogenous primer for second strand synthesis. As such,instead of having the promoter in the oligo-T primer, the promoter cannow be included in the sequence of the primer for second strandsynthesis, thereby reversing the direction of transcription. Forexamples of various means that have been described for producing alibrary of sense RNA as an amplified product, see U.S. PatentApplication No. 20040161741; Goff et al., 2004 BMC Genomics 5; 76-84;and Marko et al., 2005 BMC Genomics 6; 27-39; the contents of all ofwhich are incorporated by reference.

Labeling of this sense RNA does not produce, however, a productcompatible with monophasic arrays that are exclusively designed tohybridize with labeled anti-sense nucleic acids. As such, it wassuggested in the aforementioned U.S. Application No. 20040161741 thatinstead of using monophasic arrays that were complementary to antisenseRNA products, the array could be designed for sequences complementary tothe original sense mRNA. On the other hand, arrays designed for use withantisense RNA products have been used with amplification processes thatgenerate sense oriented strands by the simple expedient of applying thesame solution that was originally used with the unamplified mRNA, i.e.,the sense amplification product was used as a template for synthesizinglabeled cDNA. It should be pointed out that this reverse transcriptionstep is not necessary when a biphasic array is used that has bothstrands present at each site or when a small number of commerciallyavailable arrays that have oligonucleotide targets from one strand atsome locations and targets from the other strand in other locations(Checklt arrays for example, available from Telechem International, Inc.Sunnyvale, Calif., product literature incorporated by reference herein).In these cases, some of the targets on the arrays are compatible witheither labeled sense or anti-sense products.

It should also be pointed out that a monophasic array synthesized witholigonucleotides in the anti-sense orientation has recently becomecommercially available (the Human Exon 1.0 ST Array from Affymetrix,inc. Santa Clara, Calif., product literature incorporated by referenceherein). This array was designed by taking exon and EST sequences andusing them to design complementary sequences for the array. Kits thathave been designed to generate label these are either designed toproduce sense strand cDNA products (WT cDNA synthesis and amplificationkit, Affymetrix, Santa Clara, Calif.; product literature incorporated byreference) or designed to synthesize both labeled sense and antisenseproducts.

When carrying out RNA profiling studies, the limiting amount of nucleicacids in a sample is not the only concern. First, when carrying outstudies on transformed cell lines or tumors, there will often besufficient material for direct methods of CGH analysis to identifyamplifications or deletions of chromosomal content. On the other hand,other specimens may be very small (biopsies or microdissected material)or of low quality (archival biopsy specimens). Second, for the purposesof prognostic diagnosis of cancer, it is often critical to identifychromosomal aberrations prior to there being a significant physicalappearance in a tumor. For instance, gross level changes in copy numberof the human telomerase gene have been identified in Pap smears bycomparative FISH analysis and correlated with predictions of developmentof cervical carcinoma (Heselmeyer-Haddad et al, 2005 Am J Path 166;1229-1238, incorporated by reference herein). For the above reasons,numerous methods have been described in the literature for generalamplification of chromosomal DNA sequences. For a review of a number ofsystems used for this purpose see Hughes et al., 2004 Progress inBiophysics and Molecular Biology 88; 173-189, the contents of which areincorporated by reference.

It is easily understood that when doing CGH studies, both strands arepresent in equal amounts. RNA profiling studies are often carried out,however, on a basic assumption of asymmetry, i.e., when the activity ofa particular gene is being studied by means of an oligonucleotide array,it is sufficient to have sequences present from only one strand. What issometimes if not often overlooked is that transcription is notcompletely relegated to one strand, even when a single gene isconsidered. A well-recognized natural phenomenon termed anti-senseregulation takes place in cells where transcription of sequences thatare complementary to protein coding mRNA is used by cells to regulatethe amount of gene products that are made from the mRNA transcripts.Recent studies that have involved more precise measurement of the extentof sense and anti-sense sequences being transcribed from the same genehave shown that it is possible that more than twenty percent (20%) oftranscribed genes have anti-sense counterparts (Chen et al., 2004 Nucl.Acids Res. 32; 4812-4820, incorporated by reference herein).

For studies where both sense and anti-sense poly A mRNA are amplified inan asymmetric manner, the product will still consist of both [+] and [−]strands. For instance, when the Eberwine procedure is used, the mRNAtranscript in a sample will generate complementary aRNA strands. In asimilar fashion, anti-sense transcripts with polyA ends that may also bepresent in the sample will likewise be amplified and the complementarystrands generated from these templates will comprise sense mRNAsequences. In studies that have used monophasic oligonucleotide arraysfor RNA profiling, this duality has been for the most part ignored sinceonly the labeled aRNA amplification products generated signals byhybridizing to the mRNA derived sequences on the array. Expression ofanti-sense poly A sequences was not measured in such experiments due toa lack of complementary sequences on the arrays and only changes in mRNAtranscription were recognized in these studies. On the other hand,separate assessments for amplified mRNA products and antisense RNAproducts can be achieved by providing arrays with oligonucleotides thatare complementary to each orientation. The foregoing analyticaltechniques have been used for labeled unamplified RNA samples (Kumar etal., 2002 Nature Biotechnology 20; 58-63; Kampa et al., 2002 GenomeResearch 14; 331-342, both of which are incorporated by reference).

Even arrays that comprise a single orientation may be confounded by thepresence of both sense and antisense sequences in a biological samplewhen methods of amplification are used that are symmetric in nature. Anexample of this is the SMART PCR method (Clontech, Mountain View,Calif., product literature incorporated by reference herein), where bothmRNA and anti-sense transcripts serve as templates for PCR amplificationas long as they have poly A tails. Labeled products made by this processwill hybridize to a monophasic array regardless of whether the originaltemplate was a sense mRNA or an anti-sense transcript. Under theseconditions, this process was not aimed at measuring mRNA transcriptionlevels per se, but rather in measuring the overall gene activity wherecontributions from both sense and anti-sense transcripts in a samplecontribute to the ultimate signal. Similarly, in array systems whereboth strands are present at each site of the array, signals aregenerated not only by amplification products of mRNA transcripttemplates, but also by the amplification products from antisensetranscript templates regardless of whether an asymmetric or symmetricamplification process is used. In essence, these arrays also provide ameasurement of an overall gene activity without distinguishing whetherthe signal is derived from copying either sense or anti-sense transcripttemplates.

When there are broad changes in species that are represented in highnumbers in a sample, effects are easily ascertained. Only a certainpercentage of the population is, however, sufficiently represented suchthat the products are capable of generating a detectable signal, i.e.,targets that may be present in small numbers cannot be reliably detectedabove the background levels of the array. The number of targets that canbe detected as compared to the number of potential targets is frequentlyreferred to as the “call rate.” As one gets closer to background levels,random fluctuations in signal strength become more problematic, evenwith detectable signals. Furthermore, when a promoter dependentamplification method is used, there are biases involved in having thepromoter initiate transcription from sequences that were located at the3′ end or the 5′ end of the original mRNA. Thus, when a promotertranscribes from the region originally derived from the 3′ end, there isa higher representation of sequences from the 3′ region compared to the5′ end in many gene products. The converse also holds true whensequences from the 5′ region are used for the start of transcription.Thus there remains a critical need for methods that can increase thereliability of data generated from arrays as well as for methods thatcan increase the sensitivity of detection of fluctuations in copynumbers of low level target nucleic acids.

SUMMARY OF THE INVENTION

This invention provides a method of characterizing the amounts ofnucleic acids in a sample comprising the following three steps. First,there is (i) provided (a) a double-stranded library of linear nucleicacid constructs derived from said sample, wherein each constructcomprises (1) a sequence for a first RNA promoter located at one end ofthe nucleic acid construct, and (2) a sequence for a second RNA promoterlocated at the other end of the nucleic acid construct; and (b) suitablereactants for carrying out an RNA transcription reaction. In the secondstep, there are (ii) carried (a) a first transcription reaction with afirst portion of the library using the first RNA promoter to generate afirst collection of labeled nucleic acid products; and (b) a secondtranscription reaction with another portion of the library using thesecond RNA promoter to generate a second collection of labeled nucleicacid products. In the third step, (iii) hybridizing takes place between(a) the first collection to sites on a nucleic acid array, and (b) thesecond collection to sites on the nucleic acid array or to a differentnucleic acid array. In other steps, (iv) measuring the amounts ofnucleic acids hybridized to the sites; and (v) comparing the amounts tocharacterize the nucleic acids in said sample, are carried out.

In another aspect of this invention, a plus-minus determination ofnucleic acid quantities in a library can be made. This plus-minus methodfor determining the amounts of nucleic acids in a library of nucleicacids comprises the following steps. First, there are (i) generated (a)labeled [+] copies of the nucleic acids in the library; and (b) labeled[−] copies of the nucleic acids in the library. Next, (ii) hybridizationis effected between the labeled [+] copies and the labeled [−] copies toa nucleic acid array or arrays. Then, (iii) measuring the amounts ofhybridization of the labeled [+] copies and the labeled [−] copies tothe array is carried out, wherein the amounts of hybridization of thelabeled [+] copies and the amounts of hybridization of the labeled [−]copies are independently quantified, thereby determining the amounts ofthe nucleic acids.

In yet another aspect using different collections of constructs, thisinvention provides a method of determining the amounts of nucleic acidsin a library of nucleic acids. The steps of this method comprise first(i) synthesizing from the library of nucleic acids (a) a firstcollection of nucleic acid constructs comprising RNA promoters, whereintranscription from the promoters generates [+] copies of the nucleicacids; and (b) a second collection of nucleic acid constructs comprisingRNA promoters, wherein transcription from the promoters generates [−]copies of the nucleic acids. Next, there are (ii) generated (a) labeled[+] copies of the nucleic acids from the first collection; and (b)labeled [−] copies of the nucleic acids from the second collection. Inthe next step, (iii) hybridizing occurs between the labeled [+] copiesand the labeled [−] copies to a nucleic acid array or arrays. This isfollowed by (iv) measuring the amount of hybridization of the labeled[+] copies and the labeled [−] copies to the array or arrays, whereinthe amount of hybridization of the labeled [+] copies and the amount ofhybridization of the labeled [−] copies are independently quantified,thereby determining the amounts of the nucleic acids.

This invention also provides a method for determining the amounts of DNAand RNA in a library of nucleic acids. In this aspect, the methodcomprises the steps of (i) generating (a) labeled RNA copies of thenucleic acids in the library; and (b) labeled DNA copies of the nucleicacids in the library; (ii) hybridizing the RNA copies and the DNA copiesto a nucleic acid array or arrays; and (iii) measuring the amount ofhybridization of the labeled RNA copies and the labeled DNA copies tothe array or arrays, wherein the amount of hybridization of the labeledRNA copies and the amount of hybridization of the labeled DNA copies areindependently quantified, thereby determining the amounts of the nucleicacids.

This invention is also applicable to the characterization of nucleicacid amounts in a library and a reference library. In this aspect, themethod comprises the steps of (i) generating (a) labeled [+] copies ofthe nucleic acids in the library; and (b) labeled [−] copies of thenucleic acids in said library; (c) labeled [+] copies of nucleic acidsin a reference library; and (d) labeled [−] copies of the nucleic acidsin the reference library; (ii) hybridizing the labeled [+] copies andthe labeled [−] copies to one or more nucleic acid arrays; and (iii)measuring the amounts of hybridization of the labeled [+] copies and thelabeled [−] copies to the one or more arrays, wherein the amounts ofhybridization of copies (a), (b), (c) and (d) are independentlyquantified, thereby determining the amounts of the nucleic acids; and(iv) comparing said amounts of the library and the amounts of thereference library, thereby characterizing the amounts of the nucleicacids in said library.

This invention also provides a method of analyzing or characterizing alibrary of ribonucleic acids. In this method, steps are provided for (i)labeling one portion of the library of ribonucleic acids; (ii)synthesizing cDNA from a second portion of the library of ribonucleicacids; (iii) labeling the cDNA; (iv) hybridizing the labeled library ofribonucleic acids and the labeled cDNA to one or more arrays ofoligonucleotides or polynucleotides; (v) quantifying the amount ofsignal generated from the hybridized labeled library and the hybridizedlabeled cDNA, wherein the signal generated from the library isdistinguished from the signal generated by the cDNA.

The invention also provides a method of comparing expression in at leasttwo samples. In this method, there are carried out the steps ofproviding labeled [+] copies and labeled [−] copies of nucleic acids inthe samples; hybridizing the labeled [+] copies and the labeled [−]copies to one or more arrays, wherein the labeled [+] copies and thelabeled [−] copies hybridize to different array sites; and measuring theamount of hybridization on each site of the array or arrays; andcomparing the measured amounts.

Another method of characterizing the amounts of nucleic acids in asample is provided by this invention. In this method, several steps arecarried out, including the first step of (i) providing (a) first primersfor first strand synthesis and second primers for second strandsynthesis, wherein the first primers comprise a first RNA promoter andthe second primers comprise a second RNA promoter; (b) suitable reagentsfor carrying DNA polymerization reactions; and (c) suitable reagents forcarrying out RNA transcription reactions. Next, there are carried out(ii) binding the first primers to the nucleic acids in the sample andextending the first primers to form a set of first nucleic acid copies;(iii) binding the second primers to the set of first nucleic acid copiesand extending the second primers using the set of first nucleic acidcopies as templates, thereby forming a double-stranded library ofnucleic acid constructs. Then, there are carried out (iv) (a) a firsttranscription reaction with a first portion of the library using thefirst RNA promoter to generate a first collection of labeled nucleicacid products; and (b) a second transcription reaction with anotherportion of the library using the second RNA promoter to generate asecond collection of labeled nucleic acid products. This is followed by(v) hybridizing (a) the first collection to sites on a nucleic acidarray, and (b) the second collection to sites on the same nucleic acidarray or a different nucleic acid array; and (vi) measuring the amountsof nucleic acids hybridized to the sites; and (vii) comparing theamounts to characterize the nucleic acids in the sample.

Another aspect of this invention concerns a method of characterizing theamounts of nucleic acids in a sample. Several steps are carried out inthis method including a first step of (i) providing (a) first primersand third primers for first strand synthesis and second primers andfourth primers for second strand synthesis, wherein the first primerscomprise a first RNA promoter and the fourth primers comprise a secondRNA promoter; (b) suitable reagents for carrying DNA polymerizationreactions; and (c) suitable reagents for carrying out RNA transcriptionreactions. Next, there is carried out several binding reactionsincluding (ii) binding said first primers to a first portion of thenucleic acids in said sample and extending the first primers to form afirst set of first nucleic acid copies; (iii) binding the second primersto the first set of first nucleic acid copies and extending the secondprimers using the first set of first nucleic acid copies as templates,thereby forming a first double-stranded library of nucleic acidconstructs; (iv) binding said third primers to a second portion of thenucleic acids in the sample and extending said third primers to form asecond set of first nucleic acid copies; (v) binding said fourth primersto said second set of first nucleic acid copies and extending saidfourth primers using the second set of first nucleic acid copies astemplates, thereby forming a second double-stranded library of nucleicacid constructs. Then, transcription reactions (vi) are carried outincluding (vi) (a) a first transcription reaction with said firstlibrary using the first RNA promoter to generate a first collection oflabeled nucleic acid products; and (b) a second transcription reactionwith said second library using the second RNA promoter to generate asecond collection of labeled nucleic acid products. The transcriptionreactions are followed by three steps including (vii) hybridizing (a)said first collection to sites on a nucleic acid array, and (b) saidsecond collection to sites on the same nucleic acid array or a differentnucleic acid array; and (viii) measuring the amounts of nucleic acidshybridized to the sites; and (ix) comparing the amounts to characterizethe nucleic acids in said sample.

Yet another aspect provided by the present invention is a method ofdetermining the amounts of nucleic acids in a double-stranded library ofnucleic acids. Here, the steps comprise (i) generating (a) labeled [+]copies of the first strand of the nucleic acids in said library; and (b)labeled [−] copies of the first strand; (ii) hybridizing the labeled [+]copies and the labeled [−] copies to a nucleic acid array or arrays; and(iii) measuring the amounts of hybridization of the labeled [+] copiesand the labeled [−] copies to the array or arrays, wherein the amountsof hybridization of the labeled [+] copies and the amounts ofhybridization of the labeled [−] copies are independently quantified,thereby determining the amounts of the nucleic acids.

Another aspect of the present invention is a method of analyzing nucleicacids in a sample. In this method, several steps are carried outincluding a) providing RNA to be analyzed; b) adding a first sequence tothe 3′ ends of one portion of the RNA; c) binding a set of first primersto the first added sequence of the first portion, wherein the firstprimers comprise a first RNA promoter sequence; d) extending the firstprimers using the first portion of RNA as templates and generating firstcDNA copies of the first portion; e) removing the first portion RNAtemplates; and f) adding a second sequence to the 3′ ends of the firstcDNA copies of the first portion. Other steps of this method include g)binding a set of second primers to the second added sequence of thefirst cDNA copies of the first portion; h) extending the second setprimer using the first cDNA copies of the first portion as templates, toform double-stranded copies of the first portion; i) adding a thirdsequence to the 3′ ends of a second portion of the RNA; k) binding a setof third primers to the third added sequence in the second portion; l)extending the third primers using the second portion of RNA as templatesand generating first cDNA copies of the second portion; m) removing thesecond portion RNA templates; n) adding a fourth sequence to the 3′ endsof the first cDNA copies of the second portion. Other steps followincluding o) binding a set of fourth primers to the fourth addedsequence of the first cDNA copies of the second portion, wherein thefourth primers comprise a second RNA promoter sequence; p) extending theset of fourth primers using the first cDNA copies of the second portionas templates to form double-stranded copies of the second portion; q)carrying out a transcription reaction with the double-stranded copies ofthe first portion to generate labeled [−] copies of the RNA; r) carryingout a transcription reaction with the double-stranded copies of thesecond portion to generate labeled [+] copies of the RNA; and s)hybridizing the labeled [+] copies and the labeled [−] copies to anarray or arrays and separately quantifying the amount of hybridizationof the labeled [+] copies and the labeled [−] copies.

The present invention also provides a heterophasic array comprisinglabeled [+] copies of nucleic acids hybridized to the array, and labeled[−] copies of nucleic acids hybridized to the array. In this embodiment,the labeled [+] copies are separately quantifiable from the labeled [−]copies.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates various means of fragmentation of RNA prior toamplification.

FIG. 2 shows the synthesis of double-stranded constructs for generationof [+] or [−] copies of heteronuclear RNA (hnRNA).

DETAILED DESCRIPTION OF THE INVENTION Definitions

In the context of the present invention, an array is an orderedarrangement of oligonucleotides or polynucleotides fixed or immobilizedto a solid matrix, i.e., a non-porous solid support. Microarray is asynonymous term for arrays that emphasizes the size of the spaceallotted for a particular group of oligonucleotides or polynucleotideson the array. Thus, a microtitre plate and a glass slide with thousandsof target spots would both be considered to be arrays. “Bead arrays”(Michael et al., 1998 Anal Chem 70; 1242-1248) where ordering of theparticular sequences attached to beads is “decoded” (Gunderson et al.,2004 Genome Research 14; 870-877) are also considered to be covered bythe term array or array and are a subject of the present invention.

Targets in the present invention are considered to be nucleic acids thatare fixed or immobilized to an array and represent sequences ofinterest.

A nucleic acid is an oligonucleotide or polynucleotide which maycomprise natural nucleotides, modified nucleotides, nucleotide analoguesor any combination thereof. A nucleic acid may comprise homopolymeric,low complexity or high complexity sequences. A nucleic acid may beobtained by a chemical or enzymatic method or from a biological source.

The terms “sense” and “anti-sense” strands were originally derived withreference to the “sense” strand of mRNA that coded for a protein and“anti-sense” referred to its complementary sequence. In the presentinvention, these terms are retained when the present invention is beingapplied to the analysis of RNA transcripts. However, in addition tomRNA, the present invention also encompasses analysis of transcripts ofhnRNA, rRNA, snRNA or any other RNA transcript of interest. Thuspolarity is defined with reference to sequences that are identical totranscripts ([+] polarity) and complementary to transcripts ([−]polarity). Thus in the present invention, [+] labeled copies are eitherthe original target strand that has been labeled or a copy has beenprepared that comprises identical sequences. Contrariwise, [−] labeledcopies are copies that comprise sequences complementary to the originaltemplate. The present invention is also applicable to analysis of DNA.Non-limiting examples of techniques where this may be applied includedComparative Genomic Hybridization (CGH) and methylation patternanalysis. In this particular context, the designation of [+] and [−] areof a more arbitrary nature and the terms [+] polarity and [−] polarityemphasize the complementarity of the strands rather than descriptions offunctionality inherent in such terms as “sense” and “anti-sense”.

A monophasic array is an array where the majority of the targets on thearray are represented by a single polarity, i.e either a [+] polaritymonophasic array or a [−] polarity monophasic array.

A biphasic array is an array where the majority of the targets on thearray are represented by sequences derived from each strand; i.e. both[+] polarity and [−] polarity for the target are at the same site on thearray.

A heterophasic array is an array where both [+] and [−] targets arerepresented for a target of interest, but the [+] and [−] targetsequences are at separate spots or locations.

The present invention discloses novel methods and compositions where theidentification and quantification of a particular nucleic acid in asample is represented by hybridization of both a labeled [+] strand copyof the nucleic acid and a labeled [−] strand copy of the nucleic acid toan array. The [+] strand copy may be the original copy of the nucleicacid itself and the [−] sample may a complementary copy that has beensynthesized using a similar [+] strand copy as a template. On the otherhand, both [+] and [−] copies may be the result of amplificationprocesses that have created numerous copies that comprise sequences thateither comprise the same sequence ([+] copies) or a complementarysequence ([−] copies) of the original nucleic acid template.Quantification of [+] copies compared to [−] copies may be carried outsimultaneously, sequentially or in parallel.

In the present invention each target sequence in a library can beindependently represented by both [+] and [−] copies where the amount ofeach type of copy is separately determined by hybridization to an array.In some embodiments of the present invention, both [+] and [−] copiescan be generated by the same construct, for example by a double-strandedDNA molecule with a promoter at each end, where transcription from onepromoter provides [+} copies and transcription from the other endprovides [−] copies. In other embodiments of the present invention,separate constructs are made where one construct provides [+] copies andthe other construct provides [−] copies. With a construct with promotersat each end, a pool can be divided up such that one promoter is used inone set of reactions and the other promoter is used for separate set ofreactions. This is especially useful when a different marker is intendedto indicate each orientation. On the other hand, it is also possible tocarry out transcription simultaneously when each orientation has thesame marker and separation of signals is carried out by the identity ofthe polarity of the target at a given site on the array.

The present invention may be applied to a number of nucleic acidscomprising but not limited to RNA from eukaryotic sources such as poly AmRNA or heterologous RNA, RNA from prokaryotic sources, RNA from viralsources, genomic DNA from prokaryotic, eukaryotic or viral sources andartificially synthesized nucleic acids that are a product of one or morenucleic acid copying or amplification reactions.

A large number of RNA profiling studies have had sufficient materialthat an amplification step was unnecessary. Accordingly, in prior art acommon method of producing labeled products for arrays has been thesimple step of synthesis of cDNA copies, i.e., the products were in theanti-sense orientation with respect to the original mRNA. Quantificationof the original mRNA population was then carried out by measuring theamount of hybridization between the labeled anti-sense cDNA probes and acollection of sense strand targets on a array. As such, each sample gaveonly one piece of information per target site.

On the other hand, the lack of a sufficient amount of nucleic acids togenerate information on levels of specific nucleic acids in some sampleshas led to the development of a number of different systems for globalamplification of nucleic acids. These can be either of a symmetricnature where sequences from each strand are amplified or they can be ofan asymmetric nature where one strand of a nucleic acid is preferablyamplified.

When using asymmetric means of amplification that employ an RNApromoter, the labeled product has been either the RNA product itself ora labeled cDNA derived from it. In either case, a single orientation ofthe labeled samples was selected for use with the array. As describedpreviously, since the principle use of this method has been forgeneration of RNA profiles, the nucleic acid amplification products havebeen [−] strand copies of the original mRNA that were then hybridized toeither biphasic arrays or [+] strand monophasic arrays. With either typeof array, a single piece of information has been gained for each nucleicacid sequence being quantified.

With regard to symmetrical means of amplification that have used a PCRtype of reaction, even when the initial template comprised sequencesfrom only one strand, the amplification resulted in a library ofamplified nucleic acids that comprised sequences from each strand.Similar to the situation with the asymmetric amplification technologies,the labeled products from symmetric amplification have then beenhybridized either to a monophasic array that has targets derived from[+] strands or they have been used with biphasic arrays with both [+]and [−] strands at the same target site; in the latter case, the amountof signal contributed from each labeled strand is combined into a singlesignal for each target site and thus, indistinguishable from each other.

In comparative genomic hybridization (CGH) analysis, both strands areusually labeled and hybridized to an array. However, since the originaltargets (genomic DNA) comprise both strands in equal amounts and themethods used for either labeling or amplification in CGH should be equalin efficiency for each strand, both strands are usually labeled asprobes and both strands are present in the same site on a CGH array,i.e., a biphasic array. Because the signal generated in a CGH assay hasbeen the product of both labeled strands hybridizing to the same site,the particular level of signal strength derived from each strand cannotbe distinguished from each other. There has also been an increase in theutilization of oligonucleotide arrays for CGH analysis. (For a recentreview, see Yistra et al., 2006 34; 445-450, hereby incorporated byreference). Such arrays have been monophasic arrays, however, where thepresence of sequences from only one strand has been deemed necessary andsufficient to quantify genomic targets.

In summary, only a single piece of information has been obtained for atarget sequence in prior art. This has been regardless of whether thetarget was derived from nucleic acids such as DNA that contain bothstrands or derived from nucleic acids such as poly A RNA that mostlycomprises sequences from only one strand. It has also held true forproducts that have been amplified by asymmetric means such as by an RNApromoter or by symmetric means delivered by a PCR type of amplification.

In contrast to these methods, the present invention discloses novelcompositions, methods and assays whose products provide additionalinformation by determining separately and independently the level ofhybridization that can take place with hybridization of a library oflabeled [+] and [−] copies of a nucleic acid of interest to arrays. Itshould be pointed out that this is not the same as previous referencesto measurements of sense and anti-sense poly A transcripts derived froma biological sample. In those studies, the sense and anti-sensetranscripts were different nucleic acid species that were independentlytranscribed from different promoters in vivo. They also were notcompletely contiguous to each other and would comprise only partialoverlaps due to differences in initiation, termination and processing ofintrons. Again, as was described for other prior art, only a singleorientation of the strand was used for measurement for each of the invivo generated species (sense and anti-sense RNA). That is to say,measurements of the amount of in vivo sense Poly A RNA was performed byhybridization of the labeled sense mRNA to a [−] monophasic array andmeasurements of the amount of in vivo antisense RNA was measured byhybridization of the labeled antisense labeled RNA to a [+] monophasicarray. As such, a single measurement was generated for each in vivospecies. In contrast, the present invention discloses making andseparately detecting a [+] and a [−] copy of a sense mRNA with at leastone of these orientations being generated in vitro. Furthermore, thepresent invention also discloses generating and detecting labeled [+]and [−] copies of anti-sense RNA thereby providing two separatemeasurements for the amount of each initial species of interest.

By carrying out the method of the present invention, two differentmeasurements are independently collected for each nucleic acid sequenceof interest, thereby serving as a validation on performance of thesystem. Thus, when low signals are achieved due to a minorityrepresentation within a population, a coincidence of a low signal thatis slightly above background from individual measurements of bothlabeled [+] and [−] strands gives an indication that these are truesignals rather than background effects. The present invention may alsobe employed when analyzing apparent differences in samples whereindependent assessments of hybridization differences by each strand willallow the user to distinguish between an intrinsic difference in thesample and a difference derived from an experimental artifact. It hasbeen previously noted that the greatest variability in results come fromnucleic acid sequences that are present in low numbers and/or exhibitsubtle differences in amounts (Nygaard et al., 2005 BMC Genomics 6;147). Furthermore, when amplification systems are used that areasymmetric in nature, the independent use of each strand as a labeledprobe can in principle help diminish strand bias effects, i.e.,differential representation of sequences from the 5′ and 3′ ends.

Any of the various labeling and/or amplification methods that have beenpreviously described in the art for carrying out array analysis ofnucleic acids may be used for carrying out the present invention. Forinstance, labeled nucleic acids used for CGH analysis will usuallycomprise equal amounts of each labeled strand. These can be hybridizedto a heterophasic array where for a given target sequence, there will bea [+] sequence at one location and a complementary [−] sequence at aseparate location. If DNA from a test sample is labeled with a firstmarker and DNA from a control sample is labeled with a second marker,hybridization to the heterophasic array will allow two separatetest/control ratios to be generated from the first and second markerreadings; i.e. a first/second marker ratio for each [+] loci and afirst/second marker ratios for each [−] loci, thereby providing separateassessments of changes in genomic copy levels. In this case 4 datapoints would be generated for each sequence of interest ([+] and [−]copies of the test sample nucleic acids and [+] and [−] copies of thecontrol sample nucleic acids). In the present invention, any means thatare able to label, copy or amplify genomic DNA may find use with thepresent invention. These can include methods that have been described inthe past as well as methods that may be disclosed in the future. Thesalient point is not with regard to how labeled genomic DNA sequencesare generated but the use with such labeled species are made, i.e.,information is independently obtained for each strand.

As described previously by others, unamplified mRNA has been used as atemplate to generate labeled cDNA (a collection of [−] copies) and thenhybridized to an array of sense oriented nucleic acids to ascertain anmRNA profile for a sample when there are sufficient amounts of sampleavailable. In addition, we have previously cited examples (Kumar et al.,2002; Kampa et al., 2002) where the mRNA itself was directly labeled andused as a probe (a collection of [+] copies). In contrast to this priorart which is limited to a single orientation, the present inventionprovides both labeled [+] and [−] copies, thereby doubling theinformation regardless of whether the information is conveyed by DNA,RNA, or a combination of the two. In addition to the methods that havebeen described in the references cited previously for directly labelingmRNA, two other non-limiting examples that may be used for this purposeare chemically labeling the mRNA (U.S. Pat. No. 6,262,252 and Hoevel etal., 1999 Biotechniques 27; 1064-1067) or using the poly A region of themRNA as a Universal Detection Target (UDT) for hybridization of labeledoligo-T as described in U.S. Patent Application No. 20040161741. Thecontents of the foregoing publications and patent application is herebyincorporated by reference. To carry out the present invention, theselabeled libraries could be hybridized to two different monophasic arrayswhere one monophasic array has [+] orientation nucleic acids and asecond monophasic array has [−] orientation nucleic acids. Signals maybe separated by carrying out individual hybridizations with eachlibrary, i.e. hybridizing the library of [+] copies to a [+]/[−] set ofmonophasic arrays and the library of [−] copies to a separate set ofmonophasic arrays. On the other hand, both the [+] library and the [−]library may be hybridized and quantified together if there are differentsignals associated with each library. A biphasic array, such as thosederived from plasmid or PCR products may also be used in this aspect ofthe present invention. In this case, providing information from whetherthe signal is derived from the labeled poly A RNA or from the labeledcDNA copy can be carried out either by using separate biphasic arraysfor each library or using different labels for each library. Asdiscussed above, the presence of both [+] and [−] strands for eachtarget on a biphasic array implies that the signal from the labeled polyA RNA at each site on a biphasic array will be a compilation of bothsense and anti-sense poly A transcripts that were in the original sampleand reflects a gene activity measure. Signals from the labeled cDNAcopies will also have combined signals derived from the original senseand anti-sense poly A species thereby generating overall gene activitymeasurements.

Lastly, it should be mentioned that there is a less commonly used typeof array (which we have defined as a heterophasic array) that comprisessequences from both sense and antisense strands, but each orientation isfixed to a separate location. In essence, it could be considered to be acombination of putting the features of a [+] monophasic array and a [−]monophasic array onto a single array. The advantage of a heterophasicarray is that it is able to serve as a collector for strands in eitherorientation like a biphasic array, but it has the capability ofseparating signals for hybridization of each strand rather thanco-mingling them as seen with the biphasic array. A commerciallyavailable example of this type of array is the Checkit array describedearlier. When using a heterophasic array and an experimental sample hasthe [+] strand copies labeled differently from the [−] strand copies,information of the particular source of the labeled nucleic acid strandshybridized to a each site on the array may be derived from both thedifference in signal type, as well as by predetermined information onwhether it is a [+] or [−] strand target sequence at that location.Thus, independent data points can be obtained from both [+] and [−]labeled poly A RNA as well as [+] and [−] labeled cDNA copies.

The use of a heterophasic array with the present invention also allows,however, the same label to be used for both [+] and [−] labeledlibraries hybridized to the same array where differentiation betweensignals from the [+] and [−] labeled copies is carried out strictly bythe particular location on the array. This method would be of especialuse in cases where the heterophasic array has been designed to reducerepresentation of sequences that are shared by both sense and anti-sensetranscripts. Alternatively, it may also be used without suchconsiderations when the particular strand origin is not of importance(i.e., in CGH experiments or assays of overall gene activity) or if inthe context of the experiment, the signal contributions from in vivoderived antisense transcripts are considered to be of minimal nature.This method finds utility in the context of experiments where directcomparisons can be made on a heterophasic array with a sample from anexperimental condition having a first label and a normal (control)sample having a second label. Thus, a control sample can have both [+]and [−] strand products labeled with a first label and a test sample canhave both [+] and [−] strand products labeled with a second label andall four pools can be hybridized simultaneously to a single heterophasicarray. Interpretation of the nature of the signal at each site allowsindependent assessment of each labeled product. It should be pointed outthat in the foregoing example, the four pools would comprise aheterogeneous mixture that would include labeled [+] RNA strands as wellas labeled [−] cDNA strands being hybridized to an array under a singleset of conditions. It is believed, however, that under the conditionsnormally used for hybridization to arrays, the potential differencesbetween hybridization stability or efficiency for RNA compared to cDNAbinding to the array should be of a minimal nature.

Any of the methods that have been previously described in the literaturefor amplification of the original nucleic acids in samples may also finduse with the present invention. When symmetric amplification systemssuch as PCR are used, the resultant samples can be differentiallylabeled in each strand by incorporating different labels into eachprimer. One means for carrying this out has been described in U.S.Patent Application No. 20040161741 (incorporated by reference herein)where Universal Detection Targets (UDT's) are included into thesequences of primers used for amplification. For instance, mRNA can beamplified by synthesizing a collection of cDNAs using an oligo T primerwith an arbitrary selected sequence (UDT 1) appended to it. This canthen be used as a template by random primers that have a differentdiscrete sequence at their 5′ ends (UDT 2) to generate 2^(nd) strandcopies. Consequently, PCR amplification can then be carried out by usingthe UDT 1 and UDT 2 sequences as primers where UDT 1 is labeled with Cy3 and the UDT 2 is labeled with Cy5. The amplified PCR product couldthen be hybridized to a heterophasic array, a biphasic array or separatemonophasic [+] and [−] arrays and each strand of the PCR productindividually identified and quantified. Alternatively, the samples maybe used with the same label on each strand if they are hybridized to aset of positive and negative monophasic arrays or if they are hybridizedto a heterophasic array with each strand located on the same array butin different locations. In this context, labeling can be carried out byincorporating a labeled nucleotide during the amplification reaction.Again, as described previously, when two samples are being compared, aseparate label can be used for each sample. Identification of the samplesource (sample 1 or sample 2) is carried out by the particular labeldetected and the source of the strand is carried out by the location ofthe signal (on the positive or negative array when using monophasicarrays and at the positive or negative location for a heterophasicarray).

Asymmetric amplifications such as those that employ phage promoters tocarry out RNA transcription based amplification may also find use withthe present invention. Thus, as described previously, either [+] or [−]RNA may be generated depending upon whether the promoter is part of theprimer used for first strand or second strand synthesis. When oneparticular orientation is chosen for transcription, this RNA product maybe labeled and as described previously, a complementary strand may alsobe synthesized and labeled while generating cDNA copies. It is alsocontemplated that dividing pools into reactions that will make either[+] or [−] may be performed. Thus, one amplification reaction can becarried out that involves a promoter being part of the first strandprimer and a second separate amplification reaction can be carried outwith a promoter being part of the primer used for second strandsynthesis. Examples of a number of methods for incorporating a promoteror other desirable nucleic acid sequence into the second strand havebeen disclosed in U.S. Patent Application No. 20060057583 and U.S.Patent Application No. 20040161741, contents of both incorporated byreference herein. The transcript products for each of these reactionswill result in a [−] labeled RNA library and a [+] labeled RNA library,respectively. An advantage of this approach is that it results in acomparison where RNA is compared to RNA as opposed to the example citedearlier which entailed a cDNA vs RNA comparison.

Alternatively, dual promoter constructs may be used where there is apromoter at each end of a construct thereby allowing both [+] and [−]strands to be made from the same construct. This approach will minimizethe amount of reagents needed for amplification prior to thetranscription reactions and avoid dividing the sample into two separatepools at an early stage. If desired, the promoters in a dual promoterconstruct may be different from each other so that the same set ofconstructs can be used to generate a collection of labeled [+] copies inone reaction and a set of labeled [−] copies in another reaction. Thiswould be especially useful when separate labels are desired for eachstrand product where the nucleic constructs can be divided into twopools where one promoter would use one RNA polymerase to synthesize [+]copies from one end of the constructs and in a separate reaction, adifferent polymerase is used to synthesize [−] copies from the other endof the constructs. On the other hand, the promoters at each end may bethe same promoter when an array is intended to be used that willquantify labeled [+] and [−] nucleic acid copies at different locations(a heterophasic array or separate monophasic arrays) and only a singlelabel may be used for both the [+] and [−] copies. Thus, in this caseboth [+] and [−] copies can be simultaneously synthesized from a libraryof nucleic acid constructs in a single reaction. Again as describedabove, both labeled [+] and [−] products derived from one sample canhave one particular label and labeled [+] and [−] products derived froma different sample can have a second label. This approach can be of use,for example, when an experimental sample is being directly compared witha control sample.

Biphasic arrays are more common than heterophasic arrays, but twosamples can be compared on the same biphasic array by using acombination of four different labels. Thus, the dividing step that wasdescribed above can be used to create a library of nucleic acid from onesample where a first promoter is used to generate labeled [+] copieswith label 1 and a second promoter is used to generate labeled [−]copies with label 2. A second sample can then be labeled using the samepromoter systems but substituting label 3 and label 4 for the [+] and[−] copies. Thus as long as labels 1, 2, 3 and 4 are distinguishablefrom each other, a biphasic array can be used to individually quantifyeach labeled product of two samples on a single array. Separatelylabeled nucleotides may be used or modified nucleotides with chemicallyreactive groups may be incorporated, thereby allowing the addition ofany desirable label in a post-synthetic reaction. An example of thismethod is a transcription reaction with allylamine modified UTP followedby addition of an NHS ester of a dye.

One particular consideration in carrying out the method of the presentinvention is that even when mRNA is being used as a source, it is notcompletely asymmetric in nature. A fine-detailed mapping of the amountand source of polyA RNA was analyzed by labeling mRNA at the 3′ end andhybridizing it to a set of [+] and [−] monophasic arrays that almostcompletely covered chromosomes 21 and 22 (Kampa et al., 2004, GenomeResearch 14; 331-342, incorporated by reference herein). This analysisdemonstrated that in two cell lines, 11% of the “transfrags” thatoverlapped known exon, mRNA and EST sequences, were actually in theantisense orientation with regard to these sequences. Also, aspreviously discussed, some estimates of the number of genes thatgenerate anti-sense as well as sense mRNA have been estimated to be ashigh as 20%. This indicates that in any preparation of poly A RNA,labeling of anti-sense derived products is always going to be carriedout to some degree in addition to mRNA.

As such, when poly A is being analyzed, it may be desirable to haveasymmetry artificially added. One way to carry this out is in the designof the arrays, where only sequences are chosen that are known to lackantisense expression. Alternatively, if targets are present in the arraythat are transcribed as both sense as well as anti-sense and this is asubject of interest, asymmetry can be provided in the labeling reactionswhere one label will be associated with identical copies of the originaltarget sequences and a second label will be associated withcomplementary copies of the original target sequences. If oneheterophasic array is used for a sample and a second heterophasic arrayis used for a control, each label on an array can be compared to theother array to independently identify changes in both sense mRNA andanti-sense transcripts.

On the other hand, when symmetric means are being used foramplification, for example RNA samples with the SMART PCR system or DNAsamples for CGH studies, both strands are indiscriminately amplified atthe same time and an overall assessment of nucleic acid levels is madefor each nucleic acid of interest and one marker can be assigned to thetest sample and another indicator can be assigned to the control sample.In this case, the signals on an array will represent a summation ofsignals from both strands in the case of DNA and from both mRNA andantisense RNA in the case of RNA profiling. As described above, an arraycan be designed such that it detects only the segments of mRNA that havelittle or no complementary representation of antisense RNA. Furthermore,by the use of a system for labeling or amplification that makes use ofpolyA tails, only anti-sense that also has undergone poly A additionwill be generating signals. Lastly, in a method where hnRNA as well asmRNA are quantified, signals are not limited to poly A mRNA andexpression in general will be surveyed. In any of the cases cited above,the use of a pair of [+] and [−] monophasic arrays or a heterophasicarray can be used to double the information received for each sequenceof interest compared to the amount of information achieved previouslywith a biphasic array or a [+] monophasic array.

It should also be pointed out that the nature of the anti-sensetranscription might not always be relevant in methods where both mRNAand anti-sense transcripts become labeled. In the first place, theanti-sense transcripts are not usually complete complementary copies ofmRNA; they will usually have their own start and stop signals fortranscription. As such an oligonucleotide array can be designed thatthat comprises only sequences that are absent in anti-sense transcripts.Secondly, for many target sequences the amount of anti-sense RNAtranscription may be negligible compared to the amount of mRNAtranscription.

The studies that were cited previously were not so much concerned aboutthe particular levels of anti-sense so much as discerning whether therewas any detectable level of anti-sense transcription for the chromosomalloci being analyzed.

In addition to the previously described arrays that have target specificsequences in specific locations on matrices, there are arrays that useslightly different processes which eventually can generate theequivalent of the more standard monophasic and heterophasic arrays. Oneexample of this type of array is a ‘bead array’ described in (Michael etal., 1998 Anal Chem 70; 1242-1248, incorporated herein by reference. Inthis system, there is no pre-ordered arrangement of sequences on aphysical matrix. Rather, a collection of separate matrices (beads) isused, where a designated sequence is assigned to each bead.Hybridization of labeled nucleic acids to each bead is followed byrandomly sorting out of the beads onto a physical matrix such that eachexperiment ends up with a unique arrangement of beads making up anarray. At this point they are more like a classical array except thatthe particular sequence is not predetermined as in a normal array butrather there is a decoding step that is used to identify the particularsequence that eventually ended up at each site. (For a fullerdescription of this last point, refer to “Decoding Randomly ordered DNAArrays”, Gunderson et al., 2004 Genome Research 14; 870-877,incorporated by reference herein). In this particular system, theselection of sequences determines whether the array is equivalent to astandard monophasic or heterophasic array, i.e., for a given nucleicacid of interest, only one strand is represented on the beads (similarto a monophasic array) or contrariwise there may be some beads havingsequences from one strand and other beads having sequences derived fromthe complementary strand (similar to a heterophasic array). It should bepointed out that in the latter case, it would be preferred that whenboth strands for a given target are used, it would be best that they bederived from different portions of the nucleic acid target sequence toavoid self hybridization between beads. Another solution would be tocarry out parallel experiments where one set of beads comprises oneorientation and a second set of beads comprises a set of complementarysequences.

Another variant that can be used in the present invention are what aresometimes termed “universal arrays.” These are arrays that carry acollection of sequences that are deliberately selected as lacking anycomplements in the mammalian genome even though their major applicationis for identification or quantification of mammalian nucleic acidsequences. This seemingly paradoxical choice is dictated by the use ofthese arrays to provide a single array that can detect labeled nucleicacids having any particular sequences chosen by a user, thereby avoidingthe necessity of acquiring custom designed arrays. For a description ofarrays that have been used for this method see Gerry et al, 1999 J Mol.Biol. 292; 251-262, Shoemaker et al., 1996 Nature Genetics 14; 450-456,Gharizadeh et al., 2003 Nucl. Acids Res. 31; e146, all of which areincorporated by reference; they are also commercially available as“GenFlex™ TAG arrays” (Affymetrix, Inc. Santa Clara, Calif.) and “Tm100Universal Arrays” (Tm Biosciences, Toronto, Ontario, Canada). Theessential feature of these arrays is that they are used in conjunctionwith custom designed “adapter’ oligonucleotides that have two portions,a first portion comprising sequences that are complementary to one ofthe sequences that is fixed or immobilized to a particular site on theuniversal array and a second portion having a sequence that iscomplementary to a sequence that is of interest to the user.

The adapter molecule can be used for what is some times referred to asSNP (Single Nucleotide Polymorphism) analysis. In one method, theadapter is used as a primer with a polynucleotide of interest as atemplate, where incorporation or lack of incorporation of a label can beused to indicate the presence of a particular base at the SNP site thatis being queried (Fan et al., 2000 Genome research 10; 853-860,incorporated by reference herein) by hybridization of the primer to theuniversal array to quantify the amount of primers that have acquired alabel. They have also been used in ligation based assays where theligation of the adapter to a labeled second oligonucleotide will bedependent upon the identity of the nucleotide at a SNP site (Gerry etal., 1999 J Mol Bio 292; 251-262, incorporated by reference herein).

Alternatively, the adapter can serve as a bridge between a labelednucleic acid of interest and the artificial sequence on the universalarray. In essence, these universal arrays could act as sandwichhybridization assays that will localize the labeled sequence of interestto a chosen site on the universal array. Examples of commerciallyavailable arrays of this type are “GenFlex™ TAG arrays” and “Tm100Universal Arrays” cited above.

By choosing a set of sequences that are complementary to only one strandof a desirable target, a Universal Array can be transformed into amonophasic array. On the other hand, selection of set of sequences thatare derived from each strand of nucleic acid sequence of interestgenerates can be used to transform a universal array into a heterophasicarray.

In another aspect of the present invention, bacterial or heterologousRNA are quantified by the novel methods disclosed above. Additionalsteps need to be taken for these RNA targets since they are eithercompletely (bacterial mRNA) or mostly (hnRNA) missing poly A tails. Withregard to hnRNA, a further consideration is the potentially enormoussize of the RNA transcripts. The use of hnRNA could provide a morecomplete description of transcriptional activity by allowing assessmentsto be made on introns that are later excised during mRNA maturation aswell as RNA species that are non-polyadenylated, a type of RNA that maybe involved in control functions.

Due to its large size the use of hnRNA as a target would preferablyinvolve a fragmentation step. This may take place a) prior to makingcopies of the hnRNA, b) as part of as process for making copies or c)after copies have been made. Methods for fragmenting hnRNA are wellknown n the art. For instance, fragmentation of hnRNA can take place byi) physical, ii) chemical or iii) enzymatic means. Examples ofmechanical means can include but not be limited to shearing andsonication. Examples of chemical means can include but not be limited tomild alkali treatment with or without metal ions being present. Kits forcarrying out this approach are available from a number of commercialsources. Examples of enzymatic means can include but not be limited tonucleases or RNases. Examples of Rnases that may be used for thispurpose can include but not be limited to RNase III which uses secondarystructure sites as substrates and RNaseH which can digest hnRNA at siteswhere there is a complementary DNA sequence hybridiszed to the RNAstrand. With regard to the latter method the complementary nucleic acidscould be random DNA oligonucleotides or they could comprise chimericoligonucleotides with at least one segment for generating a an RNA/DNAsubstrate and one or more segments that do not provide a substrate forRNase digestion but do provide stability for binding the chimericoligonucleotide to the RNA. A description of this method has beenpreviously described in U.S. Patent Application Serial No. 20050137388(incorporated by reference herein) and is also illustrated in FIG. 1.After fragmentation, a UDE can be attached to the 5′ ends, the 3′ endsor both 3′ and 5′ ends of the fragmented RNA to provide promotersequences, primer binding sites or a combination of the foregoing tocarry out amplification reactions. Examples of methods that may be usedfor carrying out this step can include but not be limited to ligationand extension methods disclosed in U.S. Patent Application Serial No.20040161741, U.S. Patent Application Serial No. 20060057583 and U.S.Patent Application Serial No. 20050137388, all of which are incorporatedby reference.

The fragmented RNA with a UDT incorporated into the 3′ ends may be usedas a substrate for first strand cDNA synthesis, thereby generating DNAstrands with defined sequences at their 5′ ends. If desired,unfragmented RNA may also be used as a substrate for cDNA synthesis.Methods that may useful for this purpose can include but not be limitedto random priming with the unfragmented RNA as a template or by additionof a UDT to the 3′ ends of the unfragmented RNA and using the addedsequences as primer binding sites. With regard to the latter method,appendage of a UDT to the 3′ end creates a strong bias towards the 3′end sequences and for very large RNA templates, few if any cDNA copieswill completely reach the 5′ ends. As such, if using unfragmented RNA,it would be preferred to use the random primer method, where the cDNAproduct should comprise a collection of fragments whose size and 5′ endlocations depend upon the particular random sites on the RNA templatewhere a primer was bound and extended. This method would also allow theincorporation of UDT's into the 5′ ends of the cDNA's by the use ofprimers that had random 3′ ends but had a defined sequence (a UDT) atthe 5′ end. The UDT in the 5′ end could provide a primer binding site, apromoter or a combination of the foregoing in the cDNA products.

Once first strand cDNA has been synthesized from the RNA templates, theseries of steps that would subsequently be carried out should be similarto those described for poly A RNA. These can include any of the methodspreviously described in the literature as well as the previously citedU.S. Patent Application Serial No. 20040161741, U.S. Patent ApplicationSerial No. 20060057583 and U.S. Patent Application Serial No.20050137388 (all of which are incorporated by reference herein).Examples of methods that may be useful for this purpose may comprise butnot be limited to incorporation of a UDT into the 3′ ends of 1^(st)strand cDNA's by either terminal addition with TDT or the use of ablocked primer with permutational 3′ ends. Subsequent amplificationsteps can be carried out by defined cycle methods such as PCR, multiplesynthesis methods such as RNA transcription or combinations of theforegoing. Analysis of the labeled products may be carried out asdescribed above using monophasic, biphasic or heterophasic arrays.

The examples which follow are set forth to illustrate various aspects ofthe present invention but are not intended in any way to limit its scopeas more particularly set forth and defined in the claims that followthereafter.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Example 1 Analysis of BiotinLabeled [+] and [−] Copies of Poly A RNA on Monophasic Arrays afterAmplification with Single Promoter Constructs

a) A set of [+] and [−] monophasic chips can be created by taking thesequences of a commercially available [+] monophasic array andsynthesizing oligonucleotides with these sequences as well as theircomplements as a custom order from a commercial oligonucleotidesynthesis vendor. These then can be spotted onto separate arrays usingcommonly employed procedures to create complementary monophasic arrays(a set of [+] arrays and a set of [−] arrays).

b) Poly A RNA from a biological specimen can be amplified after dividingthe sample into two pools. Amplification can then be carried out witheach pool by creating first strand cDNAs by priming with an oligo Tprimer. Synthesis of the complementary second strand cDNAs can then becarried out addition of dCTP to the 3′ ends of the first strand productswith terminal transferase followed by second strand synthesis with aprimer that has a poly G segment as described in U.S. Patent ApplicationNo. 20040161741, incorporated by reference herein. For the first pool,the oligo T primer used for first strand synthesis also includessequences for a T7 RNA promoter. As such, a transcription reaction fromthe double-stranded construct will generate copies that arecomplementary to the original poly A RNA sequences. For the second pool,the primer for first strand synthesis would only contain the oligo Tsequence but the oligo G primer used for second strand cDNA synthesiswould include a T7 RNA promoter sequence. Transcripts from the secondpool of constructs will generate nucleic acids that comprise sequencesidentical to the original mRNA Poly A RNA sequences. For comparison'ssake, a similar process can be carried out with a different biologicalsample that serves as a control sample of poly A RNA. For each of thetranscription reactions, biotin-UTP (Enzo Biochem, Inc. NY) can beincluded as one of the reagents to generate biotinylated RNA products.

c) Hybridization of the pools can be carried out with the monophasicarrays described in step a).

d) The hybridized labeled products could then be detected on the arraysby using phycoerythrin attached to strepavidin (Invitrogen, Carlsbad,Calif.). It should be noted that as described previously, a sample ofpoly A RNA comprises both sense mRNA that is used for directingsynthesis of proteins and anti-sense RNA that may be involved in controlof gene expression. In this particular example, both species will bemonitored independently by hybridizing each of the pools (with [−] and[+] copies respectively) to individual [+] and [−] monophasic arrays.

For the reactions where the Oligo T/T7 primer was used for first strandsynthesis, labeled nucleic acids that are derived from mRNA in thesample (test or control) will be able hybridize to their correspondingsequences on the [+] monophasic array and nucleic acids that are derivedfrom anti-sense RNA transcripts in the specimens will hybridize to theircomplementary sequences on the [−] monophasic array. This should yieldan assessment of the amount of mRNA and anti-sense RNA in the test andcontrol samples, allowing comparisons to be made between the test sampleand the control sample for differences in both mRNA and anti-sense RNAlevels.

Contrariwise, for the test and control reactions where Oligo T was usedfor first strand synthesis and the Oligo G/T7 primer was used for secondstrand synthesis, the labeled nucleic acids derived originally from themRNA templates will hybridize to the [−] monophasic array and thelabeled RNA originally derived from the anti-sense RNA templates willhybridize to the [+] monophasic arrays. This allows an independentassessment of test/control ratios to be made that can be furthercompared to the ratios derived from the Oligo T/T7 sets of reactions.

Example 2 Analysis of Radioactively Labeled [+] and [−] Copies of Poly ARNA on Biphasic Arrays after Amplification with Single PromoterConstructs

A commercially available nylon membrane biphasic array (GF200 Human GeneFilter from Research Genetics, Huntsville Ala.) could be used instead ofthe custom designed arrays described in Example 1. This type of membraneis a biphasic array where each target locus comprises both strands. Inthis example, amplification would be carried out as described in Example1 except that ³²P labeled nucleotides would be used as the label. Afterthe amplification, the products of each of the four reactions (OligoT/T7 directed synthesis of [−] copies of the sample, Oligo G/T7 directedsynthesis of [+] copies of the sample, Oligo T/T7 directed synthesis of[−] copies of the control, Oligo G/T7 directed synthesis of [+] copiesof the control) would be hybridized to an individual nylon membrane andthen scanned and quantified using a commercial scanner (STORMPhosphorImager from Molecular Dynamics, Sunnyvale, Calif.). Due to thenature of the biphasic arrays, the amount of signal generated from bothmRNA and anti-sense templates will be summed together and serves as ameasure of gene activity, i.e., labeled [+] copies of mRNA and polyAantisense from a given genomic segment will generate signals together byhybridizing to each strand on the target sites of one filter and [−]copies of mRNA and poly A antisense from a given genomic site willgenerate, signals together by hybridizing to each strand on target sitesof a separate filter.

Example 3 Analysis of Allylamine Labeled [+] and [−] Copies of Poly ARNA on Heterophasic Arrays after PCR Amplification

A custom array would be made as described in Example 1 except that the[+] and [−] strands would be located on the same array although ondifferent sites i.e., a heterophasic array. Theoretically, commerciallyavailable arrays with some heterophasic presentation (Check-it Arraysfrom Telechem International, Sunnyvale, Calif. and 48.5 k Human HEEBOArrays from Arrays, Inc., Nashville, Tenn.) could be used for thispurpose, but each of these chips have only a limited number of targetsthat are represented by each strand and the particular choice for thesetargets were for nucleic acids that are stable in terms ofrepresentation numbers. Thus, at the present time it would be of moreuse to custom design heterophasic arrays that specifically comprisesequences that are more likely to exhibit fluctuation levels withvariations in genetic and environmental conditions. Such sequences maybe chosen as described previously in Example 1.

Amplification could be carried out by using the RNA as a single pool foramplification and an artificial UDT could be included at the 5′ end ofthe Oligo T primer used for first strand synthesis as described in U.S.Patent Application No. 20040161741 and U.S. patent application Ser. No.10/693,481, contents of both incorporated herein by reference. Althoughthere are a variety of methods disclosed in these patent applicationsthat could be used for this purpose, in this example the enzyme TdT willbe used to add a short stretch of dC's to the 3′ end of the first cDNAstrand. For second strand cDNA synthesis, an oligonucleotide that isblocked at the 3′ end and has an oligo G sequence as well as a T7promoter sequence will be hybridized to the oligo C UDT on the firststrand. Although the blocked oligo cannot be extended itself, it canserve as a template for further extension of the first cDNA strand,thereby incorporating the T7 promoter sequence into the 3′ end of thefirst cDNA strand. A series of PCR amplification could then be carriedout using a primer with the promoter sequence for one strand and aprimer with the UDT sequence as a primer for the other strand. Thisprocess could be carried out in parallel with a control sample of RNAfor comparison with the results of the test RNA sample.

In the next step, the PCR amplicons from sample and control reactionswould be divided into two pools. One pool from the sample PCR reactionwould be used for a transcription reaction that includes allylamine UTPto produce labeled RNA. The other pool from the sample PCR reactionwould use normal ribonucleotides in a transcription reaction and theunlabeled RNA products would subsequently be used as templates for arevere transcription reaction that includes allylamine dUTP. Thus, bothallylamine labeled [+] RNA and allylamine labeled [−] DNA strands willhave been generated from the same original sample. This would also holdtrue for pools made in the same way from the control PCR reaction. Byusing allylamine in this example, one would minimize differences thatmay occur due to different affinities for labeled nucleotides by RNApolymerase compared to reverse transcriptase. Post-synthetically, theallylamine labeled products could then be modified by attaching the NHSester of a Cy3 type dye (Enzo Life Sciences, Farmingdale, N.Y.).

The [+] RNA and [−] DNA from the sample source can be hybridized to oneheterophasic array and the [+] RNA and [−] DNA from the control sourcecould be hybridized to a separate heterophasic array. It should be notedthat even though both strands are present, the concentratedoligonucleotides on the array should be driving the hybridization andthere should be limited hybridization between the RNA and DNA in theliquid phase. This principle is also used in CGH experiments where bothlabeled strands are always used together. On a further note, unlabeledDNA from the double stranded PCR amplicon should be present innegligible levels compared to the labeled RNA and the labeled cDNA dueto the high level of amplification of RNA from each amplicon promoter.

Example 4 Analysis of Digoxygenin Labeled [+] and [−] Copies of Poly ARNA on Heterophasic Arrays after SMART PCR Amplification

In this example, the commercially available SMART cDNA synthesis systemwill be used (Atlas™ SMART™ Fluorescent Probe Amplification Kit,Clontech Laboratories, Mountain View, Calif., product literatureincorporated herein by reference) for test samples and control samplesof RNA. During the PCR amplification step, digoxygenin dUTP will beincluded as a label during the amplification step. Thus, both [+] and[−] strands will be labeled simultaneously for each sample.

Hybridization will be carried out with heterophasic arrays similar tothe ones described in Example 4 with the sample and control beinghybridized to separate arrays, i.e., the test sample product withlabeled [+] and [−] strands will be hybridized to one heterophasic arrayand the control sample product with labeled [+] and [−] strands will behybridized to a second heterophasic array. Detection of the extent ofhybridization could be then carried out by using the Applied BiosystemsChemiluminescence Detection Kit (Applied Biosystems, Foster City,Calif.) and the Typhoon 9410 Imager (GE Healthcare, Piscataway, N.J.).

Example 5 Analysis of Fluorescein Labeled [+] and [−] Copies of Poly ARNA from a Test Sample and Texas Red Labeled [+] and [−] Copies of PolyA RNA from a Control Sample on Heterophasic Arrays after SMART PCRAmplification

Amplification can be carried out in parallel for a test sample and acontrol sample using the SMART PCR system described above. In thisexample, however, the test sample will be labeled with FI-dUTP (EnzoLife Sciences, Farmingdale, N.Y.) and the control sample will be labeledwith the aphenylic Texas Red (TR)-dUTP described in U.S. PatentApplication 20030225247, filed Mar. 12, 2002, the contents of whichhereby incorporated by reference. Since the test sample and controlsample are now spectrally different from each other, they may besimultaneously hybridized to a single array. In this example, themonophasic arrays described previously in Example 1 will be used.Analysis of the array should generate ratios of test sample compared tocontrol samples. In contrast to the prior art, however, two separateratio assessments for each nucleic acid of interest can be achieved: FIand TR labeled [+] strands hybridized to their complementary sequenceson the [−] monophasic array to give one ratio and FI and TR labeled [−]strands hybridized to their complementary sequences on the [+]monophasic array to independently give a second ratio.

Example 6 Analysis of Cy3 Labeled [+] Copies of Poly A RNA from a TestSample and a Control Sample and Cy 5 Labeled [−] Copies of Poly A RNAfrom a Test Sample and a Control Sample on Biphasic Arrays after SMARTPCR AmplificationDual Label, Using a Transcription Based Modification ofthe SMART Amplification System and Hybridizing to Biphasic Array

A variant of the SMART process is also commercially available thatemploys transcription as well as PCR amplification (BD SMART™ mRNAAmplification kit, Clontech Laboratories, Mountain View, Calif.). Acombination of using transcription as well as PCR for the SMART systemhas previously been described in the literature by Gustincich et al.2004, Proc. Nat. Acad. Sci. USA 101; 5069-5074 and Ji et al., 2004Analyt Biochem 331; 329-339. This method could be adopted for use withthe present invention by substituting a primer for poly A tails thatincludes oligo-T linked to an SP6 promoter for one end and use the SMARTprimer with a T7 promoter that is included in the kit for the other end.After PCR amplification of a test sample and a control sample by theSMART method, the products can be divided into two separate pools. Afirst pool for each sample can be used with transcription by T7 in thepresence of allylamine-UTP for both the test sample and the controlsample to generate allylamine labeled [+] copies. In parallel, a secondpool can be used with transcription from the SP6 promoter to generateallylamine labeled [−] copies from the test sample and the controlsample. After completion of the reactions, the allylamine labelednucleic acids can be reacted with appropriate NHS esters to generatefluorescently labeled RNA products. In the case of the T7 generatedtranscripts, the NHS ester can be Cy3 and the collections of SP6transcripts can be labeled with Cy5, both dyes being available from GEHealthcare (Piscataway, N.J.). The Cy3 and Cy5 labeled RNA from thecontrol can be mixed together and hybridized to one biphasic array andthe Cy3 and Cy5 labeled RNA from the test sample can be mixed togetherand hybridized to another biphasic array. Detection of Cy3 will allowassessment of the amount of labeled [+] copies binding to theircomplementary sequences on the biphasic arrays, thus allowing generationof a test/control ratio by comparison of the Cy3 signals from thecontrol and test biphasic arrays at each locus. Detection of Cy5 willalso allow, however, assessment of the amount of labeled [−] strandsbinding to their complementary sequences on the biphasic arrays, therebyallowing generation of a separate test/control ratio. Also, it should bepointed out that due to the design of this particular example,assessments of mRNA and anti-sense transcripts are combined into asingle signal and overall gene activity is being measured and compared.

Example 7 Comparative Genomic Hybridization (CGH) Using RNATranscription Amplification and a Heterophasic Array

This particular Example is directed towards detection of amplificationof chromosomal DNA segments of chromosome arm 3q, an event that has beenassociated with tumor development (Heselmeyer-Haddad et al. 2005 Am J.Path. 166; 1229-1238). The initial part of the procedure of this Examplehas been described by Klein et al. (1999, Proc. Nat. Acad. Sci. USA 96;4494-4499). Briefly, chromosomal DNA from a sample to be tested fordevelopment of cervical carcinoma and a control sample are each digestedwith Mse I to give fragments ranging from 100 to 1,500 base pairs. T4DNA ligase is used to add an adapter (UDT) to each end of the fragmentsthat allows a single primer to be used in a subsequent PCR reaction. Forthis particular example, a variation will be used where the adaptercomprises a T7 RNA polymerase promoter segment and linear amplificationis carried out rather than PCR as described in Klein et al. (1999).After removing unligated adapters, a transcription reaction can becarried out with allylamine UTP for both the test sample and the controlsample. Although, transcription will be taking place concurrently fromeach end, it has been previously shown that there is surprisingly littleinterference between the two reactions taking place on the same doublestranded fragment and dual T7 promoter vectors are commonly used forgeneration of high yields of siRNA (Wang et al., 2000 JBC 275;40174-40179 and Wickstead et al. Mol Biochem Parasit. 125; 211-216).After synthesis, the products could be heat denatured rendering themsingle-stranded and available for modification in a post transcriptionalreaction where the test sample nucleic acids can be modified by theaddition of Dylight™ 547 and the control samples with Dylight 647™,active esters of dyes that are available from Pierce Biotechnology,Rockford, Ill.

In this particular example, a defined area of human chromosome arm 3qwill be used to design a series of sequences taken from both strands todetect amplification events. The particular sequences for the areas mostclosely identified with amplification events are located at segmentscoding for the Human Telomerase Gene (TERC) (Heselmeyer-Haddad et al.,2005 Am J Path 166; 1229-1238). After choosing appropriate sequences,custom arrays can be ordered from Nimblegen, Inc. (Madison, Wis.) forcreation of a heterophasic CGH array. Hybridization and detection of theCy3 and Cy5 nucleic acids can be carried out to determine Cy3/Cy5 ratiosfor both [+] and [−] strands.

Example 8 Analysis of Labeled [+] and [−] Copies of Poly A RNA from aTest Sample and Labeled [+] and [−] Copies of Poly A RNA from a ControlSample after Amplification with Single Promoter Constructs and UsingHeterophasic Arrays and 4 Different Markers

The RNA from a test sample and a control sample could each be divided upinto separate pools to give a total of 4 pools. As described in Example1, a T7 promoter sequence can be used as part of a primer for eitherfirst strand or second strand cDNA synthesis and the amplificationprocedure followed basically as described in Example 1. In this example,however, allylamine-UTP will be used as the label for each separatetranscription reaction and a different dye will be attached to theallylamine labeled products in the 4 pools to indicate source (test orcontrol) as well as orientation [+] or [−]. A variety of dyes that arespectrally distinct from each other may be used for this purpose. Inthis example four dyes will be used that have previously been used forsequencing with labeled ddNTP's (Lee et al., 1992 Nucleic Acids Research20; 2471-2483): 6FAM (520 nm), 5ZOE (540 nm), 5-R6G (555 nm) and NAN2(585 nm).

In Summary: Pool A=

Test sample; Oligo-T/T7 promoter primer; 6FAM marker

Pool B=

Test sample; T7 promoter in second strand primer; 5ZOE marker

Pool C=

Control sample; Oligo-T/T7 promoter primer; 5-R6G marker

Pool D=

Control sample; T7 promoter in second strand primer; NAN2 markerHybridization can then be carried out simultaneously with all four poolsto a single biphasic array. In the same way that four individual dyescan be used in sequencing to indicate the presence of a particular base,each of the dyes now represents a particular sample.

Example 9 Analysis of Labeled [+] and [−] Copies of Poly A RNA from aTest Sample and Labeled [+] and [−] Copies of Poly A RNA from a ControlSample after Amplification with Single Promoter Constructs inConjunction with Heterophasic Arrays and 4 Different Markers

To give wider separation between signals from different labels, aheterophasic chip may also be used with two samples (test and control)that are labeled with four distinct chromophores. In this way,hybridization to the array can result in having only two spectrallydistinguishable labels present at each site as opposed to the biphasicarray in Example 8, which had four different labels potentially presenton each site. By these means, spectral overlap can be minimized as afactor. The amplification and labeling could be carried out as describedin Example 8. 6FAM (520 nm) labeled Pool A and 5-R6G (555 nm) labeledPool C products that were derived from mRNA templates would bothhybridize to the [+] sites of the heterophasic array with a peakseparation of 35 nm between Pool A and Pool C signals; labeled Pool Aand C products that were derived from antisense products would hybridizeto the [−] sites of the heterophasic array. Conversely, 5ZOE (540 nm)labeled Pool B and NAN2 (585 nm) labeled Pool D products that werederived from mRNA templates would both hybridize to the [−] strand sitesof the heterophasic array with a peak separation of 45 nm between Pool Band D signals; labeled Pool B and D products that were derived fromantisense products would hybridize to the [+] sites of the heterophasicarray. These results would give two independent assessments of theamounts of mRNA and antisense poly A transcripts present in the test andcontrol samples.

Example 10 Analysis of Labeled [+] and [−] Copies of Poly A RNA from 3Test Samples and Labeled [+] and [−] Copies of Poly A RNA from a ControlSample after Amplification with Single Promoter Constructs inConjunction with Heterophasic Arrays and 4 Different Markers

RNA samples from a control and three test samples can be amplifiedindividually and used with 4 different dyes to indicate their source.First strand synthesis can be carried out with an oligo T primer togenerate cDNA strands from polyA mRNA. After removal of the RNAtemplates, a ligation can be carried out with the permutationaloligonucleotides described in U.S. Patent Application 20060057583. Foreach sample, a ligation reaction can be carried out with one set ofpartially double stranded adapter molecules that will add a uniquesequence to the ends of 50% of the cDNA strands. After removal of theunligated material, a second set of partially double-stranded adaptermolecules can be ligated that will add the unique sequence to theremaining 50% of the cDNA. For the purposes of this example, the uniquesequence will be a T7 promoter sequence. The reactions can then bedivided up into two pools and labeling carried out essentially asdescribed in Example 3. A first pool will be used in a transcriptionreaction with allylamine UTP. These will generate copies that areessentially the same sequence as the original nucleic acid strands. Foreach source a different dye can be attached to the allylamine moietiesin the RNA: Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 594 and AlexaFluor 647 (Invitrogen, Carlsbad, Calif.). Hybridization to aheterophasic array will allow assessments of the amount of sense andanti-sense present for each sample where the particular fluor willindicate the sample source. The second pool will be used in atranscription reaction with normal nucleotides and then used withreverse transcriptase and allylamine dUTP to generate labeled cDNAcopies. Again, the same colored dyes may be used for each samplefollowed by hybridization to a heterophasic array where quantificationof sense and anti-sense transcripts can be compared with the resultsobtained with the labeled RNA from the first pool.

Example 11 Bead Array, Single Label

As described earlier, bead arrays are a form of array that may also beused with the present invention. Due to the design of this particularsystem, the identity of the particular sequence associates with a beadcan be arbitrarily assigned a particular code. Thus a sequence derivedfrom one strand (the [+] sequence, for instance) of a target sequence ofinterest can have one particular code and a sequence from the otherstrand (the [−] sequence) can have a different code. Thus, functionallyspeaking, after beads are sorted out to make up an array, it essentiallycomprises a biphasic array where information on the particular locationsof both [+] sequence and [−] sequence beads are decoded afterhybridization. In addition for coding for the particular sequence on thebead, however, this system can also be used to assign the particularsource of a labeled nucleic acid.

Thus, in the present example, the method of amplification in Example 1can be carried out where a pool of constructs is made that can be usedto generate biotin labeled [+] copies. These can be hybridized to beadswith both [+] and [−] versions of the sequences of interest, where thecode describes both the identity of the target and its orientation. Asecond pool made as described in Example 1, can then be used to generatebiotin labeled [−] copies. These can be hybridized to a different groupof beads, where the coding indicates not only the sequence andorientation, but also that the beads were hybridized to the second pool.This process can be carried out further if the two pools above are madefrom an experimental sample and they are to be compared to a control.Thus a further step is taken where the control sample is used to make apool that generates [+] copies (a third pool) and a pool that generates[−] copies (a fourth pool). The third and fourth pools are thenhybridized to a third and fourth group of beads respectively, where thecoding can be used to indicate sequence, orientation and pool source.After the groups are each hybridized, the beads may be mixed togetherand sorted out followed by a decoding step. Even though each pool hasthe same label, the decoding step indicates the particular pool that wasused to generate labeled copies as well as the amount from each poolthat hybridizes to [+] and [−] beads. Thus both [+] copies and [−]copies are independently used to indicate the amount of mRNA and poly Aantisense that was present in the experimental sample and the controlsample.

By assigning different decoding numbers for control and sample, they canbe distinguished from each other even though the hybridized nucleicacids have the same label. Similarly, with regard to the sequences onthe beads, both [+] and [−] sequences for a particular target can beassigned different decoding numbers and again the same label can be usedfor both the [+] and the [−] strands. Hybridize separately and thendistinguish afterwards through decoding whether it was the testhybridization reaction or the control reaction and whether it is the [+]strand on the bead or the [−] sequence on the bead.

Example 12 TAG Array, Single Label

As described previously, a few arrays have been described that are socalled “universal arrays” and are commercially available as “GenFlex™TAG arrays” (Affymetrix, Inc. Santa Clara, Calif.) and “Tm100 UniversalArrays” (Tm Biosciences, Toronto, Ontario, Canada). These are intendedto provide universal hybridization to any set of desirable nucleic acidsequences thereby obviating the need to make custom arrays. Thisparticular product essentially uses a variation of sandwichhybridization where adapter oligonucleotides are used that have twosegments, a first portion which is complementary to a labeled nucleicacid of interest and a second portion which is complementary to aselected sequence on the TAG array. Hybridization of the adapter to thelabeled nucleic acid allows binding of the labeled nucleic acid to aselected site on the TAG array. This method is similar to the bead arrayabove, except that a coding sequence is added that will localize atarget sequence to a selected position on the array.

Example 13 Analysis of hnRNA by a Biphasic Array

a) Preparation of Biphasic Arrays

In this example, amplification and labeling are carried out as describedin Example 6, except that sequences are added to the Universal Array toconvert it into a biphasic array for selected target sequences ofinterest.

Biphasic arrays are prepared for an entire chromosome according to themethod described in Berton et al., 2004, Science, 306, 2242-2246 wherefor each target region a sequence is located at one site on the arrayand its complement is located on a different site.

b) Preparation of Poly A RNA

Complete RNA is isolated from a control sample and an experimentalsample using the PureLink Micro-to-Midi Total RNA Purification System(Invitrgogen, Carlsbad, Calif.), and rRNA is removed by the RibosomeTranscriptome Isolation Kit (Invitrogen, Carlsbad, Calif.). Theremaining material is fragmented using the Ambion RNA fragmentation kit(Ambion, Inc., Austin, Tex.). Conversion of any 3′ Phosphate ends toHydroxyl ends is carried out by treatment with Calf IntestinalPhosphatase. PolyA Polymerase is then used to add a poly A tail to the3′ termini of the fragments. The tailed material is divided into twopools, Pool A and Pool B which will be used to generate [+] copies and[−] copies respectively.

c) First Strand Synthesis

A library of cDNA copies is made from each of the pools by hybridizingwith an Oligo T promoter. In the case of Pool B the first strand primeralso has sequences for a T7 Promoter. Extension is carried out byReverse Transcriptase to generate cDNA copies Pool A will have cDNAstrands with oligo T sequences their 5′ ends and Pool B will have cDNAstrands with oligo T-T7 Promoter sequences at their 5′ end.

d) Second Strand Synthesis

RNA templates are removed by treatment with alkali followed byneutralization. Blocked Primers are allowed to hybridize to the 3′ endsof the cDNA copies as described in U.S. Patent Application No.20060057583. For Pool A, the Blocked Primers used in this step will havea T7 promoter sequence followed by a set of N permutational nucleotides.For Pool B, the Blocked Primers will have an Arbitrary Primer BindingSequence followed by a set of N permutational nucleotides. Since the 3′ends of the cDNAs represent a random set of sequences, the use of thepermutational set allows binding events to take place between theBlocked Primers and the 3′ ends of the cDNA's followed by extension ofthe 3′ end of the cDNA's thereby incorporating sequences derived fromthe T7 Promoter (Pool A) or the arbitrary primer binding sequence (PoolB). This is similar to random primer synthesis except that it is thetemplate that undergoes extension instead of the primer. The BlockedPrimers are subsequently denatured from their templates and removed fromthe reaction mixtures. New primers are then added that are capable ofextension (i.e., they aren't blocked). In the case of Pool A, theseprimers comprise the T7 promoter sequence and for Pool B, the complementto the Arbitrary Primer Binding Sequence. Strand extension results in adouble set of double-stranded nucleic acid constructs each having asingle promoter located at one end. In the case of Pool A, transcriptionin the presence of allylamine UTP will generate labeled [+] copies ofsequences of the original hnRNA and in the case of Pool B, transcriptionwill generate allyamine labeled [−] copies, i.e., sequencescomplementary to the original sequences of the hnRNA. Because of thenumber of steps it has taken to reach this point, FIG. 1 presents anillustration of the sequence of the steps described above.

e) Preparation of Fluorescently Labeled Material from Pool A

Post synthetic labeling of Pool A is carried out with Cy3 NHS ester forlabeling the experimental sample and Cy5 NHS ester for the controlsample.

f) Preparation of Labeled Material from Pool B

Post synthetic labeling of Pool B is carried out with Cy5 NHS ester forlabeling the experimental sample and Cy5 NHS ester for the controlsample.

g) Hybridization to Biphasic Arrays

Pool A from the experimental and control samples (Cy3 and Cy5respectively) are hybridized to a biphasic array and Pool B from theexperimental and control samples (Cy5 and Cy3 respectively) arehybridized to a separate biphasic array and each spot analyzed for theamount of Cy3 and Cy5 signals and the ratios compared for each array.

Many obvious variations will no doubt be suggested to those of ordinaryskill in the art in light of the above detailed description and examplesof the present invention. All such variations are fully embraced by thescope and spirit of the invention as more particularly defined in theclaims that now follow.

1. A method of characterizing the amounts of nucleic acids in a samplecomprising the steps of (i) providing (a) a double-stranded library oflinear nucleic acid constructs derived from said sample, wherein eachconstruct comprises: (1) a sequence for a first RNA promoter located atone end of said nucleic acid construct, and (2) a sequence for a secondRNA promoter located at the other end of said nucleic acid construct;and (b) suitable reactants for carrying out an RNA transcriptionreaction; (ii) carrying out (a) a first transcription reaction with afirst portion of said library using said first RNA promoter to generatea first collection of labeled nucleic acid products; and (b) a secondtranscription reaction with another portion of said library using saidsecond RNA promoter to generate a second collection of labeled nucleicacid products; (iii) hybridizing (a) said first collection to sites on anucleic acid array, and (b) said second collection to sites on the samenucleic acid array or a different nucleic acid array; and (iv) measuringthe amounts of nucleic acids hybridized to said sites; and (v) comparingsaid amounts to characterize the nucleic acids in said sample.
 2. Themethod of claim 1, wherein said first RNA promoter and said second RNApromoter are different promoters.
 3. The method of claim 1, wherein saidfirst RNA promoter and said second RNA promoter are the same promoterand said first transcription reaction and said second transcriptionreaction are the same reaction.
 4. The method of claim 2, wherein saidfirst RNA promoter comprises a T7 RNA promoter sequence, a T3 promotersequence or an SP6 promoter sequence, and said second RNA promotercomprises a T7 RNA promoter sequence, a T3 promoter sequence or an SP6promoter sequence.
 5. The method of claim 3, wherein said first RNApromoter and said second RNA promoter comprise a T7 RNA promoter, a T3RNA promoter or an SP6 RNA promoter.
 6. The method of claim 1, whereinsaid first collection is labeled during said first transcriptionreaction and said second collection is labeled during said secondtranscription reaction.
 7. The method of claim 1, wherein said firstcollection is labeled after said first transcription reaction and saidsecond collection is labeled after said second transcription reaction.8. The method of claim 1 wherein said first collection is labeled duringsaid first transcription reaction and said second collection is labeledafter said second transcription reaction.
 9. The method of claims 6 or8, wherein labeling during said transcription reactions is carried outby incorporating a modified nucleotide during said transcriptionreaction.
 10. The method of claim 9, wherein said modification comprisesa chemically reactive group and after said transcription reaction achemical reaction is carried out that adds a label to said modifiednucleotide through said chemically reactive group.
 11. The method ofclaims 6, 7 or 8, wherein after said transcription reaction, a labelingreaction is carried out by means of a chemical linkage of a label to anunmodified nucleotide.
 12. The method of claim 1 wherein the label ofsaid first collection and the label of said second collection comprisethe same label.
 13. The method of claim 1, wherein the label of saidfirst collection and the label of said second collection comprisedifferent labels.
 14. The method of claim 1, wherein said firstcollection and said second collection are hybridized to the same array.15. The method of claim 1, wherein said first collection and said secondcollection are hybridized to different arrays.
 16. The method of claim1, wherein said array or arrays comprise a monophasic array, biphasicarray or a heterophasic array.
 17. The method of claim 1, wherein saidlabel comprises a fluorescent compound, a phosphorescent compound, achemiluminescent compound, a chelating compound, an electron densecompound, a magnetic compound, an intercalating compound, an energytransfer compound and a combination of any of the foregoing.
 18. Themethod of claim 1, wherein said double-stranded library of nucleic acidconstructs is a first library and said method further comprises thesteps of: (vi) providing (a) a second double-stranded library of linearnucleic acid constructs wherein each construct comprises: (1) a sequencefor a first RNA promoter located at one end of said linear nucleic acidconstruct and a sequence for a (2) second RNA promoter located at theother end of said linear nucleic acid construct; (vii) carrying out (a)a third transcription reaction with a first portion of said secondlibrary using said first RNA promoter to generate a third collection oflabeled nucleic acid products; and (b) a fourth transcription reactionwith another portion of said second library using said second RNApromoter to generate a fourth collection of labeled nucleic acidproducts; (viii) hybridizing said third collection to sites on a nucleicacid array and hybridizing the fourth collection to sites on said arrayor on a different nucleic acid array.
 19. The method of claim 1, furthercomprising carrying out steps (i) through (v) with a second sample andcomparing the amounts of nucleic acids in said samples.
 20. A method ofdetermining the amounts of nucleic acids in a library of nucleic acidscomprising the steps of (i) generating (a) labeled [+] copies of saidnucleic acids in said library; and (b) labeled [−] copies of saidnucleic acids in said library; (ii) hybridizing said labeled [+] copiesand said labeled [−] copies to a nucleic acid array or arrays; and (iii)measuring the amounts of hybridization of said labeled [+] copies andsaid labeled [−] copies to said array, wherein the amounts ofhybridization of said labeled [+] copies and the amounts ofhybridization of said labeled [−] copies are independently quantified,thereby determining the amounts of said nucleic acids.
 21. The method ofclaim 20, wherein said labeled [+] copies and said labeled [−] copiesare generated by transcription from a collection of linear nucleic acidconstructs derived from said library, wherein a sequence for a first RNApromoter is located at one end of a linear nucleic acid construct andsequence for a second RNA promoter is located at the other end of saidlinear nucleic acid construct and wherein said labeled [+] copies aregenerated by transcribing from said first promoter and said labeled [−]copies are generated by transcribing from said second promoter.
 22. Themethod of claim 20, wherein said labeled [+] copies and said labeled [−]copies are generated by transcription from a collection of linearnucleic acid constructs derived from said library, wherein a firstportion of said linear nucleic acid constructs comprises a first RNApromoter directing transcription of said [+] copies and wherein a secondportion of said linear nucleic acid constructs comprises a second RNApromoter directing transcription of said [−] copies.
 23. The method ofclaim 20, wherein said labeled [+] copies comprises RNA and said labeled[−] copies comprises DNA.
 24. The method of claim 20, wherein saidlabeled [−] copies comprise RNA and said labeled [+] copies compriseDNA.
 25. The method of claim 20, wherein said labeled [+] copiescomprises RNA and said labeled [−] copies comprises RNA.
 26. The methodof claim 20, wherein said labeled [−] copies comprise RNA and saidlabeled [+] copies comprise RNA.
 27. The method of claim 20, whereinsaid labeled [+] copies and said labeled [−] copies are synthesized froma collection of linear nucleic acid constructs derived from saidlibrary, wherein asymmetric PCR is used to amplify one strand of saidlinear nucleic acid constructs to provide said labeled [+] copies andasymmetric PCR is used to amplify the other strand of said linearnucleic acid constructs to provide said labeled [−] copies.
 28. Themethod of claim 21 or 22, wherein said first RNA promoter and saidsecond RNA promoter are the same promoter.
 29. The method of claim 21 or22, wherein said first RNA promoter and said second RNA promoter aredifferent promoters.
 30. The method of claim 28, wherein said firstpromoter and said second promoter comprise a T7 RNA promoter, a T3promoter and an SP6 promoter.
 31. The method of claim 28, wherein saidfirst RNA promoter comprises a T7 RNA promoter sequence, a T3 promotersequence or an SP6 promoter sequence, and said second RNA promotercomprises a T7 RNA promoter sequence, a T3 promoter sequence or an SP6promoter sequence.
 32. The method of claim 20, wherein said array orarrays comprise monophasic arrays, biphasic arrays or heterophasicarrays.
 33. The method of claim 20, wherein said label comprises afluorescent compound, a phosphorescent compound, a chemiluminescentcompound, a chelating compound, an electron dense compound, a magneticcompound, an intercalating compound, an energy transfer compound and acombination of any of the foregoing.
 34. The method of claim 20, whereinthe nucleic acids in said library comprise DNA.
 35. The method of claim20, wherein the nucleic acids in said library comprise RNA.
 36. Themethod of claim 35, wherein said RNA comprises hnRNA or mRNA, or both.37. The method of claim 36, wherein said hnRNA has been fragmented. 38.A method of determining the amounts of nucleic acids in a library ofnucleic acids comprising the steps of (i) synthesizing from said libraryof nucleic acids (a) a first collection of nucleic acid constructscomprising RNA promoters, wherein transcription from said promotersgenerates [+] copies of said nucleic acids; and (b) a second collectionof nucleic acid constructs comprising RNA promoters, whereintranscription from said promoters generates [−] copies of said nucleicacids (ii) generating (a) labeled [+] copies of said nucleic acids fromsaid first collection; and (b) labeled [−] copies of said nucleic acidsfrom said second collection; (iii) hybridizing said labeled [+] copiesand said labeled [−] copies to a nucleic acid array or arrays; and (iv)measuring the amount of hybridization of said labeled [+] copies andsaid labeled [−] copies to said array or arrays, wherein the amount ofhybridization of said labeled [+] copies and the amount of hybridizationof said labeled [−] copies are independently quantified, therebydetermining the amounts of said nucleic acids.
 39. The method of claim38, wherein said RNA promoters in steps (i)(a) and (i)(b) are the samepromoter.
 40. The method of claim 38, wherein said RNA promoters insteps (i)(a) and (i)(b) are different promoters.
 41. The method of claim39, wherein said RNA promoters in steps (i)(a) and (i)(b) comprise a T7RNA promoter, a T3 promoter and an SP6 promoter.
 42. The method of claim40, wherein said RNA promoter in step (i)(a) comprises a T7 RNA promotersequence, a T3 promoter sequence or an SP6 promoter sequence, and saidRNA promoter in step (i)(b) comprises a T7 RNA promoter sequence, a T3promoter sequence or an SP6 promoter sequence.
 43. The method of claim38, wherein said array or arrays comprise monophasic arrays, biphasicarrays or heterophasic arrays.
 44. The method of claim 38, wherein saidfirst collection is labeled during said first transcription reaction andsaid second collection is labeled during said second transcriptionreaction.
 45. The method of claim 38, wherein said first collection islabeled after said first transcription reaction and said secondcollection is labeled after said second transcription reaction.
 46. Themethod of claim 38, wherein said first collection is labeled during saidfirst transcription reaction and said second collection is labeled aftersaid second transcription reaction.
 47. The method of claims 44 or 46,wherein labeling during said transcription reactions is carried out byincorporating a modified nucleotide during said transcription reaction.48. The method of claim 47, wherein said modification comprises achemically reactive group and after said transcription reaction achemical reaction is carried out that adds a label to said modifiednucleotide through said chemically reactive group.
 49. The method ofclaims 44, 45 or 46, wherein after said transcription reaction, alabeling reaction is carried out by means of a chemical linkage of alabel to an unmodified nucleotide.
 50. The method of claim 38, whereinsaid label comprises a fluorescent compound, a phosphorescent compound,a chemiluminescent compound, a chelating compound, an electron densecompound, a magnetic compound, an intercalating compound, an energytransfer compound and a combination of any of the foregoing.
 51. Amethod of determining the amounts of nucleic acids in a library ofnucleic acid comprising the steps of (i) generating (a) labeled RNAcopies of said nucleic acids in said library; and (b) labeled DNA copiesof said nucleic acids in said library; (ii) hybridizing said RNA copiesand said DNA copies to a nucleic acid array or arrays; and (iii)measuring the amount of hybridization of said labeled RNA copies andsaid labeled DNA copies to said array or arrays, wherein the amount ofhybridization of said labeled RNA copies and the amount of hybridizationof said labeled DNA copies are independently quantified, therebydetermining the amounts of said nucleic acids.
 52. The method of claim51, wherein said RNA copies comprise [+] copies of said nucleic acidsand said DNA copies comprise [−] copies of said nucleic acids.
 53. Themethod of claim 51, wherein said RNA copies comprise [−] copies of saidnucleic acids and said DNA copies comprise [+] copies of said nucleicacids.
 54. The method of claim 51, wherein the nucleic acids in saidlibrary comprise RNA and wherein said labeled RNA copies generated instep (i)(a) are derived by labeling RNA in said library.
 55. The methodof claim 51, wherein said labeled DNA copies generated in step (i)(b)are copied from RNA templates.
 56. The method of claim 51, wherein saidarray or arrays comprise monophasic arrays, biphasic arrays orheterophasic arrays.
 57. The method of claim 51, wherein said labelcomprises a fluorescent compound, a phosphorescent compound, achemiluminescent compound, a chelating compound, an electron densecompound, a magnetic compound, an intercalating compound, an energytransfer compound and a combination of any of the foregoing.
 58. Amethod of characterizing the amounts of nucleic acids in a librarycomprising the steps of (i) generating (a) labeled [+] copies of saidnucleic acids in said library; and (b) labeled [−] copies of saidnucleic acids in said library; (c) labeled [+] copies of nucleic acidsin a reference library; and (d) labeled [−] copies of said nucleic acidsin said reference library; (ii) hybridizing said labeled [+] copies andsaid labeled [−] copies to one or more nucleic acid arrays; and (iii)measuring the amounts of hybridization of said labeled [+] copies andsaid labeled [−] copies to said one or more arrays, wherein the amountsof hybridization of copies (a), (b), (c) and (d) are independentlyquantified, thereby determining the amounts of said nucleic acids; and(iv) comparing said amounts of said library and said amounts of saidreference library, thereby characterizing the amounts of said nucleicacids in said library.
 59. The method of claim 58, wherein said labeled[+] copies in steps (i)(a) and (i)(c) and said labeled [−] copies insteps (i)(b) and (i)(d) are generated by transcription from a collectionof linear nucleic acid constructs derived from said library, wherein asequence for a first RNA promoter is located at one end of a linearnucleic acid construct and sequence for a second RNA promoter is locatedat the other end of said linear nucleic acid construct and wherein saidlabeled [+] copies are generated by transcribing from said firstpromoter and said labeled [−] copies are generated by transcribing fromsaid second promoter.
 60. The method of claim 58, wherein said labeled[+] copies in steps (i)(a) and (i)(c) and said labeled [−] copies insteps (i)(b) and (i)(d) are generated by transcription from a collectionof linear nucleic acid constructs derived from said library, wherein afirst portion of said linear nucleic acid constructs comprises a firstRNA promoter directing transcription of said [+] copies and wherein asecond portion of said linear nucleic acid constructs comprises a secondRNA promoter directing transcription of said [−] copies.
 61. The methodof claim 58, wherein said [+] copies in said library and said [−] copiesin said library comprise a first label and said [+] copies in saidreference library and said [−] copies in said reference library comprisea second label.
 62. The method of claim 61, wherein said first label andsaid second label comprise the same label.
 63. The method of claim 61,wherein said first label and said second label comprise different labelsfrom each other.
 64. The method of claim 58, wherein said [+] copies insaid library, said [−] copies in said library, said [+] copies in saidreference library, and said [−] copies in said reference librarycomprise different labels from one another.
 65. The method of claim 58,wherein said array or arrays comprise monophasic arrays, biphasic arraysor heterophasic arrays.
 66. The method of claim 58, wherein said labelcomprises a fluorescent compound, a phosphorescent compound, achemiluminescent compound, a chelating compound, an electron densecompound, a magnetic compound, an intercalating compound, an energytransfer compound and a combination of any of the foregoing.
 67. Amethod of analyzing/characterizing a library of ribonucleic acidscomprising the steps of (i) labeling one portion of said library ofribonucleic acids; (ii) synthesizing cDNA from a second portion of saidlibrary of ribonucleic acids; (iii) labeling said cDNA; (iv) hybridizingsaid labeled library of ribonucleic acids and said labeled cDNA to oneor more arrays of oligonucleotides or polynucleotides; (v) quantifyingthe amount of signal generated from said hybridized labeled library andsaid hybridized labeled cDNA, wherein the signal generated from saidlibrary is distinguished from the signal generated by said cDNA.
 68. Themethod of claim 67, wherein the label used for said labeled library andthe label used for said labeled complementary library comprise the samelabel and the signals generated from said labeled library are inphysical locations that are different from the physical locations of thesignals generated from said labeled complementary library.
 69. Themethod of claim 68, wherein said array is a heterophasic array and saidlabeled library and said labeled complementary library are hybridized tothe same heterophasic array.
 70. The method of claim 68, wherein saidlabeled library and said labeled complementary library are hybridized toseparate arrays.
 71. The method of claim 70, wherein said labeledlibrary is hybridized to one or more monophasic, biphasic orheterophasic arrays and said labeled complementary library is hybridizedto one or more monophasic, biphasic or heterophasic arrays.
 72. Themethod of claim 67, wherein the label used for said labeled library andthe label used for said complementary library comprise different labels.73. The method of claim 72, wherein said labeled library and saidlabeled complementary library are hybridized to the same array.
 74. Themethod of claim 73, wherein said array is a heterophasic array or abiphasic array.
 75. The method of claim 67, wherein said label comprisesa fluorescent compound, a phosphorescent compound, a chemiluminescentcompound, a chelating compound, an electron dense compound, a magneticcompound, an intercalating compound, an energy transfer compound and acombination of any of the foregoing.
 76. A method of comparingexpression in at least two samples comprising the steps of: providinglabeled [+] copies and labeled [−] copies of nucleic acids in saidsamples; hybridizing said labeled [+] copies and said labeled [−] copiesto one or more arrays, wherein said labeled [+] copies and said labeled[−] copies hybridize to different array sites; and measuring the amountof hybridization on each site of said array or arrays; and comparing themeasured amounts.
 77. The method of claim 76, wherein said labeled [+]copies and said labeled [−] copies are generated by transcription from acollection of linear nucleic acid constructs derived from a library,wherein a sequence for a first RNA promoter is located at one end of alinear nucleic acid construct and a sequence for a second RNA promoteris located at the other end of said linear nucleic acid construct andwherein said labeled [+] copies are generated by transcribing from saidfirst promoter and said labeled [−] copies are generated by transcribingfrom said second promoter.
 78. The method of claim 76, wherein saidlabeled [+] copies and said labeled [−] copies are generated bytranscription from a collection of linear nucleic acid constructsderived from a library, wherein a first portion of said linear nucleicacid constructs comprises a first RNA promoter directing transcriptionof said [+] copies and wherein a second portion of said linear nucleicacid constructs comprises a second RNA promoter directing transcriptionof said [−] copies.
 79. The method of claims 77 or 78, wherein thenucleic acids in said library comprise DNA.
 80. The method of claims 77or 78, wherein the nucleic acids in said library comprise RNA.
 81. Themethod of claim 80, wherein said RNA comprises hnRNA or mRNA, or both.82. The method of claim 81, wherein said hnRNA has been fragmented. 83.The method of claim 76, wherein said array or arrays comprise monophasicarrays, biphasic arrays or heterophasic arrays.
 84. The method of claim76, wherein said label comprises a fluorescent compound, aphosphorescent compound, a chemiluminescent compound, a chelatingcompound, an electron dense compound, a magnetic compound, anintercalating compound, an energy transfer compound and a combination ofany of the foregoing.
 85. A method of characterizing the amounts ofnucleic acids in a sample comprising the steps of (i) providing (a)first primers for first strand synthesis and second primers for secondstrand synthesis, wherein said first primers comprise a first RNApromoter and said second primers comprise a second RNA promoter; (b)suitable reagents for carrying DNA polymerization reactions; and (c)suitable reagents for carrying out RNA transcription reactions; (ii)binding said first primers to the nucleic acids in said sample andextending said first primers to form a set of first nucleic acid copies;(iii) binding said second primers to said set of first nucleic acidcopies and extending said second primers using said set of first nucleicacid copies as templates, thereby forming a double-stranded library ofnucleic acid constructs; (iv) carrying out (a) a first transcriptionreaction with a first portion of said library using said first RNApromoter to generate a first collection of labeled nucleic acidproducts; and (b) a second transcription reaction with another portionof said library using said second RNA promoter to generate a secondcollection of labeled nucleic acid products; (v) hybridizing (a) saidfirst collection to sites on a nucleic acid array, and (b) said secondcollection to sites on the same nucleic acid array or a differentnucleic acid array; and (vi) measuring the amounts of nucleic acidshybridized to said sites; and (vii) comparing said amounts tocharacterize the nucleic acids in said sample.
 86. The method of claim85, wherein said first RNA promoter and said second RNA promoter aredifferent promoters.
 87. The method of claim 85, wherein said first RNApromoter and said second RNA promoter are the same promoter and saidfirst transcription reaction and said second transcription reaction arethe same reaction.
 88. The method of claim 86, wherein said first RNApromoter comprises a T7 RNA promoter sequence, a T3 promoter sequence oran SP6 promoter sequence, and said second RNA promoter comprises a T7RNA promoter sequence, a T3 promoter sequence or an SP6 promotersequence.
 89. The method of claim 87, wherein said first RNA promoterand said second RNA promoter comprise a T7 RNA promoter, a T3 RNApromoter or an SP6 RNA promoter.
 90. The method of claim 85, whereinsaid first collection is labeled during said first transcriptionreaction and said second collection is labeled during said secondtranscription reaction.
 91. The method of claim 85, wherein said firstcollection is labeled after said first transcription reaction and saidsecond collection is labeled after said second transcription reaction.92. The method of claim 85, wherein said first collection is labeledduring said first transcription reaction and said second collection islabeled after said second transcription reaction.
 93. The method ofclaims 90 or 92, wherein labeling during said transcription reactions iscarried out by incorporating a modified nucleotide during saidtranscription reaction.
 94. The method of claim 93, wherein saidmodification comprises a chemically reactive group and after saidtranscription reaction a chemical reaction is carried out that adds alabel to said modified nucleotide through said chemically reactivegroup.
 95. The method of claims 90, 91 or 92, wherein after saidtranscription reaction, a labeling reaction is carried out by means of achemical linkage of a label to an unmodified nucleotide.
 96. The methodof claim 85, wherein the label of said first collection and the label ofsaid second collection comprise the same label.
 97. The method of claim85, wherein the label of said first collection and the label of saidsecond collection comprise different labels.
 98. The method of claim 85,wherein said nucleic acid array and said different nucleic acid arraycomprise monophasic arrays, biphasic arrays or heterophasic arrays. 99.The method of claim 85, wherein said label comprises a fluorescentcompound, a phosphorescent compound, a chemiluminescent compound, achelating compound, an electron dense compound, a magnetic compound, anintercalating compound, an energy transfer compound and a combination ofany of the foregoing.
 100. A method of characterizing the amounts ofnucleic acids in a sample comprising the steps of (i) providing (a)first primers and third primers for first strand synthesis and secondprimers and fourth primers for second strand synthesis, wherein saidfirst primers comprise a first RNA promoter and said fourth primerscomprise a second RNA promoter; (b) suitable reagents for carrying DNApolymerization reactions; and (c) suitable reagents for carrying out RNAtranscription reactions; (ii) binding said first primers to a firstportion of the nucleic acids in said sample and extending said firstprimers to form a first set of first nucleic acid copies; (iii) bindingsaid second primers to said first set of first nucleic acid copies andextending said second primers using said first set of first nucleic acidcopies as templates, thereby forming a first double-stranded library ofnucleic acid constructs; (iv) binding said third primers to a secondportion of the nucleic acids in said sample and extending said thirdprimers to form a second set of first nucleic acid copies; (v) bindingsaid fourth primers to said second set of first nucleic acid copies andextending said fourth primers using said second set of first nucleicacid copies as templates, thereby forming a second double-strandedlibrary of nucleic acid constructs; (vi) carrying out (a) a firsttranscription reaction with said first library using said first RNApromoter to generate a first collection of labeled nucleic acidproducts; and (b) a second transcription reaction with said secondlibrary using said second RNA promoter to generate a second collectionof labeled nucleic acid products; (vii) hybridizing (a) said firstcollection to sites on a nucleic acid array, and (b) said secondcollection to sites on the same nucleic acid array or a differentnucleic acid array; (viii) measuring the amounts of nucleic acidshybridized to said sites; and (ix) comparing said amounts tocharacterize the nucleic acids in said sample.
 101. The method of claim100, wherein said first RNA promoter and said second RNA promoter aredifferent promoters.
 102. The method of claim 100, wherein said firstRNA promoter and said second RNA promoter are the same promoter and saidfirst transcription reaction and said second transcription reaction arethe same reaction.
 103. The method of claim 101, wherein said first RNApromoter comprises a T7 RNA promoter sequence, a T3 promoter sequence oran SP6 promoter sequence, and said second RNA promoter comprises a T7RNA promoter sequence, a T3 promoter sequence or an SP6 promotersequence.
 104. The method of claim 102, wherein said first RNA promoterand said second RNA promoter comprise a T7 RNA promoter, a T3 RNApromoter or an SP6 RNA promoter.
 105. The method of claim 100, whereinsaid first collection is labeled during said first transcriptionreaction and said second collection is labeled during said secondtranscription reaction.
 106. The method of claim 100, wherein said firstcollection is labeled after said first transcription reaction and saidsecond collection is labeled after said second transcription reaction.107. The method of claim 100, wherein said first collection is labeledduring said first transcription reaction and said second collection islabeled after said second transcription reaction.
 108. The method ofclaims 105 or 107, wherein labeling during said transcription reactionsis carried out by incorporating a modified nucleotide during saidtranscription reaction.
 109. The method of claim 108, wherein saidmodification comprises a chemically reactive group and after saidtranscription reaction a chemical reaction is carried out that adds alabel to said modified nucleotide through said chemically reactivegroup.
 110. The method of claims 105, 106 or 107, wherein after saidtranscription reaction, a labeling reaction is carried out by means of achemical linkage of a label to an unmodified nucleotide.
 111. The methodof claim 100, wherein the label of said first collection and the labelof said second collection comprise the same label.
 112. The method ofclaim 100, wherein the label of said first collection and the label ofsaid second collection comprise different labels.
 113. The method ofclaim 100, wherein said nucleic acid array and said different nucleicacid array comprise monophasic arrays, biphasic arrays or heterophasicarrays.
 114. The method of claim 100, wherein said label comprises afluorescent compound, a phosphorescent compound, a chemiluminescentcompound, a chelating compound, an electron dense compound, a magneticcompound, an intercalating compound, an energy transfer compound and acombination of any of the foregoing.
 115. A method of determining theamounts of nucleic acids in a library of double-stranded nucleic acidscomprising the steps of (i) generating (a) labeled [+] copies of onestrand of said double-stranded nucleic acids in said library; and (b)labeled [−] copies of said one strand; (ii) hybridizing said labeled [+]copies and said labeled [−] copies to a nucleic acid array or arrays;and (iii) measuring the amounts of hybridization of said labeled [+]copies and said labeled [−] copies to said array or arrays, wherein theamounts of hybridization of said labeled [+] copies and the amounts ofhybridization of said labeled [−] copies are independently quantified,thereby determining the amounts of said nucleic acids.
 116. The methodof claim 115, wherein said generating step (i), said labeled [+] copiesand said labeled [−] copies comprise or are derived from PCR.
 117. Themethod of claim 115, wherein said generating step (i), said labeled [+]copies and said labeled [−] copies comprise or are derived from atranscription reaction.
 118. The method of claim 115, wherein saidnucleic acid array or arrays comprise monophasic arrays, biphasic arraysor heterophasic arrays.
 119. The method of claim 115, wherein said labelcomprises a fluorescent compound, a phosphorescent compound, achemiluminescent compound, a chelating compound, an electron densecompound, a magnetic compound, an intercalating compound, an energytransfer compound and a combination of any of the foregoing.
 120. Amethod of analyzing nucleic acids in a sample comprising the steps of a)providing RNA to be analyzed; b) adding a first sequence to the 3′ endsof one portion of said RNA; c) binding a set of first primers to saidfirst added sequence of said first portion, wherein said first primerscomprise a first RNA promoter sequence; d) extending said first primersusing said first portion of RNA as templates and generating first cDNAcopies of said first portion; e) removing said first portion RNAtemplates; f) adding a second sequence to the 3′ ends of said first cDNAcopies of said first portion; g) binding a set of second primers to saidsecond added sequence of said first cDNA copies of said first portion;h) extending said second set primer using said first cDNA copies of saidfirst portion as templates, to form double-stranded copies of said firstportion; i) adding a third sequence to the 3′ ends of a second portionof said RNA; k) binding a set of third primers to said third addedsequence in said second portion; l) extending said third primers usingsaid second portion of RNA as templates and generating first cDNA copiesof said second portion; m) removing said second portion RNA templates;n) adding a fourth sequence to the 3′ ends of said first cDNA copies ofsaid second portion; o) binding a set of fourth primers to said fourthadded sequence of said first cDNA copies of said second portion, whereinsaid fourth primers comprise a second RNA promoter sequence; p)extending said set of fourth primers using said first cDNA copies ofsaid second portion as templates to form double-stranded copies of saidsecond portion; q) carrying out a transcription reaction with saiddouble-stranded copies of said first portion to generate labeled [−]copies of said RNA; r) carrying out a transcription reaction with saiddouble-stranded copies of said second portion to generate labeled [+]copies of said RNA; and s) hybridizing said labeled [+] copies and saidlabeled [−] copies to an array or arrays and separately quantifying theamount of hybridization of said labeled [+] copies and said labeled [−]copies.
 121. The method of claim 120, further comprising a step whereinsaid RNA is fragmented prior to said addition steps
 122. The method ofclaims 120 or 121, further comprising a step wherein 3′ ends that areblocked for extension are unblocked
 123. The method of claim 122,wherein said unblocking step comprises treatment with a kinase, or aphosphatase.
 124. The method of claim 121, wherein said fragmentationstep is carried out by physical, chemical or enzymatic means
 125. Themethod of claim 124, wherein said physical means comprise shearing orsonication
 126. The method of claim 124, wherein said chemical meanscomprise treatment with a solution having a pH value higher than 7.0127. The method of claim 124, wherein said enzymatic means comprisetreatment with a nuclease or an RNase
 128. The method of claim 127,wherein said nuclease comprises S1 nuclease or mung bean nuclease. 129.The method of claim 127, wherein said RNase comprises RNase Ill. 130.The method of claim 127, wherein said RNase comprises RNaseH and saidmethod further comprises a step wherein nucleic acids are annealed tosaid RNA to render the RNA as a substrate for RNase H digestion. 131.The method of claim 130, wherein said nucleic acids comprisedeoxyribonucleotides.
 132. The method of claim 130, wherein said nucleicacids further comprise ribonucleotides, nucleotide analogues or acombination thereof.
 133. The method of claim 132, wherein saidnucleotide analogues comprise peptide nucleic acids, ribonucleotideswith universal or degenerate bases, deoxyribonucleotides with universalor degenerate bases, or a combination of any of the foregoing.
 134. Themethod of claim 120, wherein one or more of said addition stepscomprises a ligation step or a polymerization step.
 135. The method ofclaim 134, wherein said ligation step is carried out by a DNA ligase oran RNA ligase.
 136. The method of claim 134, wherein said polymerizationstep is carried out by poly A polymerase
 137. The method of claim 134,wherein said polymerization is carried out by hybridizing an adapternucleic acids to the 3′ ends of said RNA and extending said RNA with aDNA polymerase wherein said adapter nucleic acids are used as templatesfor said extension
 138. The method of claim 137, wherein the 3′ ends ofsaid adapter nucleic acids have blocked 3′ ends and are incapable ofextension.
 139. The method of claim 138, wherein the 3′ ends of saidadapter nucleic acids comprise a set of permutational sequences. 140.The method of claim 120, wherein said first RNA promoter and said secondRNA promoter are the same promoter.
 141. The method of claim 120,wherein said first RNA promoter and said second RNA promoter aredifferent promoters.
 142. The method of claim 140, wherein said RNApromoter comprises a T7 RNA promoter sequence, a T3 promoter sequence oran SP6 promoter sequence.
 143. The method of claim 141, wherein saidfirst RNA promoter and said second RNA promoter are selected from thegroup consisting of a T7 RNA promoter, a T3 RNA promoter and an SP6 RNApromoter.
 144. The method of claim 120, wherein (i) said first portionand said second portion are the same portion (ii) and said steps (b)through (r) are substituted by the following steps: b′) adding a firstsequence to the 3′ ends of said RNA; c′) binding a set of first primersto said first added sequence wherein said first primers comprise a firstRNA promoter sequence; d′) extending said first primers using said RNAas templates and generating first cDNA copies of said RNA; e′) removingsaid RNA templates; f′) adding a second sequence to the 3′ ends of saidfirst cDNA copies; g) binding a set of second primers to said secondadded sequence wherein said second primers comprise a second RNApromoter sequence; h) extending said second set of primers using saidfirst cDNA copies as templates to form double-stranded copies of saidRNA; i) carrying out a transcription reaction with said double-strandedcopies of said RNA to generate labeled [−] copies of said RNA; j)carrying out a transcription reaction with said double-stranded copiesof said RNA to generate labeled [+] copies of said RNA; and k)hybridizing said labeled [+] copies and said labeled [−] copies to anarray and separately quantifying the amount of hybridization of saidlabeled [+] copies and said labeled [−] copies.
 145. The method of claim120, wherein the label of said [+] copies and the label of said [−]copies comprise the same label.
 146. The method of claim 120, whereinthe label of said [+] copies and the label of said [−] copies comprisedifferent labels.
 147. The method of claim 120, wherein said labeled [+]copies and said labeled [−] copies are hybridized to the same array orarrays.
 148. The method of claim 120, wherein said labeled [+] copiesand said labeled [−] copies are hybridized to different arrays.
 149. Themethod of claim 120, wherein said array or arrays comprise a nucleicacid target and its complement at the same site.
 150. The method ofclaim 120, wherein said array or arrays comprise a nucleic acid targetat one site and a complementary nucleic acid target at a different site.151. A heterophasic array comprising labeled [+] copies of nucleic acidshybridized to said array, and labeled [−] copies of nucleic acidshybridized to said array, wherein said [+] copies are separatelyquantifiable from said [−] copies.
 152. The heterophasic array of claim151, wherein said labeled [+] copies and said labeled [−] copiescomprise the same label.
 153. The heterophasic array of claim 151,wherein the label of said [+] copies is different from the label of said[−] copies.
 154. The heterophasic array of claim 151, wherein saidlabeled [+] copies and said labeled [−] copies are derived from a firstsample or first library, and wherein said heterophasic array compriseslabeled [+] copies and labeled [−] copies derived from a second sampleor second library.
 155. The heterophasic array of claim 151, whereinsaid labeled [+] copies comprises RNA and said labeled [−] copiescomprises DNA.
 156. The heterophasic array of claim 151, wherein saidlabeled [−] copies comprise RNA and said labeled [+] copies compriseDNA.
 157. The heterophasic array of claim 151, wherein said labeled [+]copies comprises RNA and said labeled [−] copies comprises RNA.
 158. Theheterophasic array of claim 151, wherein said labeled [−] copiescomprise RNA and said labeled [+] copies comprise RNA.
 159. Theheterophasic array of claim 151, wherein said label comprises afluorescent compound, a phosphorescent compound, a chemiluminescentcompound, a chelating compound, an electron dense compound, a magneticcompound, an intercalating compound, an energy transfer compound and acombination of any of the foregoing.